PROPYLENE SYNTHESIS USING ENGINEERED ENZYMES

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is

200206_403WO_SEQUENCE_LISTING.txt. The text file is 364 KB, was created on September 18, 2013, and is being submitted electronically via EFS-Web.

BACKGROUND

Propylene is primarily produced as a by-product of petroleum refining and of ethylene production using a steam cracking process. Propylene is separated from a mixture of hydrocarbons obtained from cracking or other refining processes by fractional distillation. Propylene is typically produced from non-renewable fossil fuels, petroleum, natural gas, and to a lesser extent coal. Propylene can also be produced in on-purpose reactions, such as propane dehydrogenation, metathesis or syngas-to-olefms plants.

Propylene is a major industrial chemical intermediate that is converted into a variety of chemicals and plastics. Manufacturers of polypropylene account for nearly two thirds of worldwide propylene demand. Polypropylene is a plastic that is used for the manufacture of films, packaging, caps, closures, and individual parts for the electrical and automotive industry. Propylene is also used to produce chemicals including acrylonitrile, oxo chemicals, propylene oxide, cumene, isopropanol, acrylic acid, butanol, and butanediol.

Currently, refinery by-product production of propylene can no longer satisfy market demand. There is a need for alternative processes for on-purpose propylene production, particularly a green process that exhibits increased safety, decreased harmful waste and emissions, and savings in cost and energy compared to petrochemically-derived propylene.

SUMMARY OF INVENTION

In one aspect, the present disclosure provides non-naturally occurring CI metabolizing organisms, wherein the non-naturally occurring CI metabolizing organisms convert a CI substrate to propylene. In certain embodiments, the CI metabolizing organism is: a non-naturally occurring CI metabolizing bacterium; a non- naturally occurring obligate C 1 metabolizing organism; a non-naturally occurring C 1 metabolizing organism, wherein the non-naturally occurring CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; a non-naturally occurring syngas utilizing bacterium that naturally possesses the ability to utilize syngas; or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO. In certain aspects, the non-naturally occurring C 1 metabolizing organisms include an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid {e.g., 4-oxalocrotonate

decarboxylase). In further aspects, the non-naturally occurring CI metabolizing organisms do not have a functional PHB synthase or a substantial amount of functional PHB synthase.

In another aspect, non-naturally occurring CI metabolizing organisms include an exogenous nucleic acid encoding a crotonyl CoA thioesterase. The non- naturally occurring CI metabolizing organism may be a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; a non-naturally occurring syngas utilizing bacterium that naturally possesses the ability to utilize syngas;or a non-naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO.

In yet another aspect, non-naturally occurring CI metabolizing organisms include an exogenous nucleic acid encoding a crotonase. In certain embodiments, the non-naturally occurring CI metabolizing organism is: a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI

metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the organism does not include Pichia pastoris; a non-naturally occurring

methanotrophic or methylotrophic bacterium; a non-naturally occurring syngas utilizing bacterium that naturally possesses the ability to utilize syngas; or a non-naturally occurring CO utilizing bacterium, wherein the CO utilizing bacterium naturally possesses the ability to utilize CO.

In another aspect, the present disclosure provides non-naturally occurring microbial organisms, wherein the non-naturally occurring microbial organisms include an exogenous nucleic acid encoding 4-oxalocrotonate decarboxylase, and wherein the non-naturally occurring microbial organisms convert a carbon substrate to propylene. In certain embodiments, the non-naturally occurring microbial organisms further include an exogenous nucleic acid encoding crotonase, or an exogenous nucleic acid encoding crotonase and an exogenous nucleic acid encoding crotonyl thioesterase. In certain embodiments, the non-naturally occurring microbial organisms do not have a functional PHB synthase or a substantial amount of functional PHB synthase.

Also disclosed herein are methods of producing propylene in non- naturally occurring organisms described herein, wherein the methods comprise culturing the non-naturally occurring organisms under conditions sufficient to produce propylene.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows an exemplary pathway for synthesis of propylene from acetyl CoA in a genetically modified organism. Enzymes and genes encoding enzymes for transformation of the identified substrates to products include: phaA, phaB, crotonase, crotonyl CoA thioesterase, and 4-oxalocrotonate decarboxylase.

FIGURES 2A-D show exemplary 4-oxalocrotonate decarboxylase (4- OD) amino acid sequences that may be used in propylene synthesis pathways as described herein.

FIGURE 3 shows exemplary crotonyl-CoA thioesterase amino acid sequences that may be used in propylene synthesis pathways as described herein.

FIGURES 4A-E show exemplary crotonase amino acid sequences that may be used in propylene synthesis pathways as described herein.

FIGURE 5 shows SDS-PAGE analysis of heterogeneously expressed 4- OD genes in E. coli. The first lane for each sample shows total cell protein and the second lane for each sample shows soluble protein following lysis and clarification. The arrow on the left shows the approximate migration of 4-OD proteins (note sequence variation causes slight changes in migration for each individual 4-OD sequence).

FIGURE 6 shows sample 4-OD activity assays run under conditions as described showing decarboxylation of 4-oxalocrotonate.

FIGURE 7 shows a sample chromatogram of propylene detected on a HP5890 GC-FID under the assay conditions as described.

DETAILED DESCRIPTION

The instant disclosure provides compositions and methods for the biocatalysis of propylene and other desirable intermediates or products from methane or other CI substrates using genetically engineered CI metabolizing organisms. The instant disclosure also provides compositions and methods for the biocatalysis of propylene from carbon substrates using novel metabolic enzyme(s) in genetically engineered microbial organisms.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

Additional definitions are set forth throughout this disclosure.

In the present description, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. The term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "have" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting. The term "comprise" means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

As used herein, the term "non-naturally occurring" when used in reference to a bacterium or an organism means that the bacterium or organism has at least one genetic alternation that is not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the bacterium or organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary enzymes include enzymes within a crotonate synthesis pathway or propylene synthesis pathway. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the non-naturally occurring organism that is altered from its naturally occurring state.

As used herein, the term "host" refers to a bacterium or organism that has not yet been genetically modified with the capability to convert a carbon substrate to crotonyl-CoA, crotonic acid, or propylene, as disclosed herein. A host C 1 metabolizing bacterium or organism is selected for transformation with at least one exogenous nucleic acid encoding an enzyme to yield a non-naturally occurring CI metabolizing bacterium or organism with the capability to convert a CI substrate to crotonyl-CoA, crotonic acid, or propylene. A host microbial organism is selected for transformation with at least one exogenous nucleic acid encoding 4-oxalocrotonate decarboxylase to yield a non-naturally occurring microbial organism with the capability to convert a carbon substrate to propylene. A host bacterium or organism may already possess other genetic modifications conferring it with desired properties, unrelated to propylene synthesis pathways and intermediates disclosed herein. For example, a host bacterium or organism may possess genetic modifications conferring high growth, tolerance of contaminants or particular culture conditions, or ability to metabolize different carbon substrates.

As used herein, the term "microbial organism," "microorganism", "microbes", or "organism" refers to any prokaryotic or eukaryotic microbial species from the domains of Archaea, Bacteria, or Eukarya. The term is intended to include prokaryotic or eukaryotic cells or organisms having microscopic size.

As used herein, the term "CI metabolizing bacterium" refers to any bacterium that has the ability to oxidize a CI compound (i.e., does not contain carbon- carbon bonds). A CI metabolizing bacterium may or may not use other carbon substrates, such as sugars and complex carbohydrates, for energy and biomass.

As used herein, the term "obligate CI metabolizing organism" refers to those organisms which exclusively use organic compounds that do not contain carbon- carbon bonds (C 1 substrate) for the generation of energy.

As used herein, the term "CI metabolizing organism" refers to any organism that has the ability to oxidize a CI substrate (i.e., does not contain carbon- carbon bonds) but may or may not use other carbon substrates, such as sugars and complex carbohydrates, for energy and biomass. A CI metabolizing organism includes bacteria, yeast (not including Pichia pastoris), and Archaea. A CI metabolizing organism includes CI metabolizing bacteria.

As used herein, the term "methanotrophic bacterium" refers to any methylotrophic bacterium that has the ability to oxidize methane as its primary carbon and energy source.

As used herein, the term "methylotrophic bacterium" refers to any bacterium capable of oxidizing organic compounds that do not contain carbon-carbon bonds. An "obligate methylotrophic bacterium" is a bacterium which is limited to the use of carbon substrates that do not contain carbon-carbon bonds for the generation of energy. Facultative methylotrophs are able to utilize multi-carbon compounds in addition to single carbon substrates.

As used herein, the term "CO utilizing bacterium" refers to a bacterium that naturally possesses the ability to oxidize carbon monoxide (CO) as a source of carbon and energy. Carbon monoxide may be utilized from "synthesis gas" or

"syngas", a mixture of carbon monoxide and hydrogen produced by gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Syngas may also include C0 ₂ , methane, and other gases in smaller quantities. CO utilizing bacterium does not include bacteria that must be genetically modified for growth on CO as its carbon source.

As used herein, the term "CI substrate" or "CI compound" refers to any carbon containing molecule or composition that lacks a carbon-carbon bond. CI substrates include methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, methyl halogens (bromomethane,

chloromethane, iodomethane, dichloromethane, etc.), or cyanide.

As used herein, the term "propylene", also known as 1-propene, propene, or methylethylene, refers to an unsaturated organic compound having the chemical formula C ₃ H ₆ and molecular mass of 42.08 g/mol. It has one double bond and is the second simplest member of the alkene class of hydrocarbons. Propylene is a gas at room temperature and atmospheric pressure. Propylene is a structural isomer of cyclopropane.

As used herein, "exogenous" means that the referenced molecule (e.g., nucleic acid) or referenced activity (e.g., enzyme activity) is introduced into the host bacterium or organism. The molecule can be introduced, for example, by introduction of a nucleic acid into the host genetic material such as by integration into a host chromosome or by introduction of a nucleic acid as non-chromosomal genetic material, such as on a plasmid. When the term is used in reference to expression of an encoding nucleic acid, it refers to introduction of the encoding nucleic acid in an expressible form into the bacterium or organism. When used in reference to an enzymatic activity, the term refers to an activity that is introduced into the host reference bacterium or organism. Therefore, the term "endogenous" or "native" refers to a referenced molecule or activity that is present in the host bacterium or organism. The term

"heterologous" refers to a molecule or activity that is derived from a source other than the referenced species or strain whereas "homologous" refers to a molecule or activity derived from the host bacterium or organism. Accordingly, a bacterium or organism comprising an exogenous nucleic acid of the invention can utilize either or both a heterologous or homologous nucleic acid.

It is understood that when more than one exogenous nucleic acid is included in a bacterium or organism that the more than one exogenous nucleic acid refers to the referenced encoding nucleic acid or enzymatic activity, as discussed above. It is also understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host bacterium or organism on separate nucleic acid molecules, on a polycistronic nucleic acid molecule, on a single nucleic acid molecule encoding a fusion protein, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein, an organism can be modified to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein (e.g., propylene pathway enzyme or protein). Where two exogenous nucleic acids encoding propylene synthesis activity are introduced into a host organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid molecule, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or enzymatic activities refers to the number of encoding nucleic acids or the number of enzymatic activities, not the number of separate nucleic acid molecules introduced into the host organism.

As used herein, "nucleic acid" refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acids include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), both of which may be single or double stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.

As used herein, "crotonic acid" or "trans-2-butenoic acid" refers to a short-chain unsaturated carboxylic acid with the formula CH ₃ CF CHCO ₂ H. As used herein, crotonic acid includes "crotonate", a salt or ester of crotonic acid.

As used herein, "4-oxalocrotonate" refers to an unsaturated carboxylic acid with the formula H0 ₂ C(C=0)CH ₂ CH=CHC0 ₂ - (keto form) or

H0 ₂ C(COH)=CHCH=CHC0 ₂ - (enol form), which interconvert spontaneously. As used herein, 4-oxalocrotonate also includes salts or esters, the equivalent molecule with the alternate acid group deprotonated, the doubly unprotonated basic form of the molecule, or the doubly protonated acid form of the molecule.

As used herein, "4-oxalocrotonate decarboxylase" or "4-OD" or "4- oxalocrotonate carboxy-lase (2-oxopent-4-enoate-forming)" refers to an enzyme family that catalyzes the decarboxylation of 4-oxalocrotonate to 2-oxopent-4-enoate and C0 ₂ . 4-OD is involved in the meta-cleavage pathway for the degradation of phenols, modified phenols, and catechols. As disclosed herein, 4-OD is used to catalyze the decarboxylation of crotonic acid to propylene and C0 ₂ . As used herein, 4-OD also encompasses mutants or variants of a native 4-OD enzyme that has reduced or eliminated decarboxylation activity on 4-oxalocrotonate, but retains or has increased decarboxylation activity on crotonic acid as a substrate. 4-OD also refers to any enzyme capable of catalyzing the decarboxylation of crotonic acid to propylene and C0 ₂ .

As used herein, "crotonyl CoA thioesterase" refers to an enzyme that catalyzes the conversion of crotonyl-CoA to crotonic acid.

As used herein, "crotonase" or "3-hydroxybutyryl-CoA dehydratase" or "enoyl-CoA hydratase" refers to an enzyme involved in the butyrate/butanol-producing pathway, which catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA.

As used herein, "β-ketothiolase" refers to an enzyme in the PHB synthesis pathway that catalyzes the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. Biosynthesis of PHB in most bacteria is initiated by β-ketothiolase.

As used herein, "acetoacetyl coenzyme A reductase" refers to an enzyme in the PHB synthesis pathway that catalyzes the reduction of acetoacetyl-CoA to 3- hydroxybutyryl-CoA.

As used herein, "PHB synthase", also known as "PHB polymerase", refers to an enzyme in the PHB synthesis pathway that converts a hydroxybutyryl-CoA monomer into polyhydroxybutyrate.

As used herein, "polyhydroxybutyrate" or "PHB" refers to a homopolymer of hydroxybutyric acid units. PHB is a type of polyhydroxyalkanoate (PHA), which is a biological polyester. Polyhydroxybutyrate is a crystalline thermoplastic synthesized by a broad range of bacteria as a form of energy storage molecule. Poly-3-hydroxybutyrate (P3HB) form is the most common type of PHB, but other PHBs include poly-4-hydroxybutytrate (P4HB), poly(3-hydroxybutyrate-co-4- hydroxybutyrate) (P3HB-co-4HB), or other copolymers. CI Metabolizing Bacteria or Organisms

A variety of C 1 metabolizing host organisms can be transformed or genetically engineered to produce a product of interest (e.g., propylene, crotonic acid, or crotonyl-CoA).

In certain embodiments, a CI metabolizing organism may be a CI metabolizing bacterium, which refers to any bacterium that has the ability to oxidize a CI compound. A CI metabolizing bacterium may also use other carbon substrates, such as sugars and complex carbohydrates, for energy and biomass. A CI metabolizing bacterium includes methanotrophic bacteria (methylotrophic bacterium that has the ability to oxidize methane as an energy source) or methylotrophic bacteria (any bacterium capable of oxidizing organic compounds that do not contain carbon-carbon bonds). Methanotrophic bacteria are classified into three groups based on their carbon assimilation pathways and internal membrane structure: type I (gamma proteobacteria), type II (alpha proteobacteria, and type X (gamma proteobacteria). Type I

methanotrophs use the ribulose monophosphate (RuMP) pathway for carbon assimilation whereas type II methanotrophs use the serine pathway. Type X

methanotrophs use the RuMP pathway but also express low levels of enzymes of the serine pathway. Methanotrophic bacteria are grouped into several genera:

Methylomonas, Methylobacter, Methylococcus, Methylocystis, Methylosinus,

Methylomicrobium, Methanomonas, and Methylocella. Exemplary methantrophic bacteria include: Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporium OB3b (NRRL B-l 1,196), Methylosinus sporium (NRRL B-l 1,197), Methylocystis parvus (NRRL B-l 1,198), Methylomonas methanica (NRRL B-l 1,199), Methylomonas albus (NRRL B-l 1,200), Methylobacter capsulatus (NRRL B-l 1,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylocella palustris (ATCC 700799), Methylocella tundrae, Methylacidiphilum infernorum, Methylibium petroleiphilum, and Methylomicrobium alcaliphilum. Methylotrophic bacteria encompass a diverse group, including both gram-negative and gram-positive genera. Methylotrophic bacteria include facultative methylotrophs (have the ability to oxidize organic compounds which do not contain carbon-carbon bonds, but may also utilize other carbon substrates such as sugars and complex carbohydrates), obligate methylotrophs (limited to the use of organic compounds that do not contain carbon- carbon bonds), and methanotrophic bacteria. Examples of methylotrophic genera include: Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium,

Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and Pseudomonas. Exemplary methylotrophic bacteria include: Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi,

Methylobacterium chloromethanicum, Methylobacterium nodularis, Methylomonas clara, Methylibium petroleiphilum, Methylobacillus flagellates, Silicibacter pomeroyi DSS-3, Burkholderia phymatum STM815, Granulibacter bethesdensis NIH1.1, and Paracoccus denitriflcans.

A CI metabolizing bacterium includes bacteria that utilize formate or cyanide or naturally possesses the ability to utilize syngas or carbon monoxide (CO) (Wu et al, 2005 PLoS Genet. I :e65; Abrini et al, 1994, Arch. Microbiol. 161 :345; WO2008/028055; Tanner et al, 1993, Int. J. Syst. Bacteriol. 43:232-236). Exemplary bacteria that naturally possesses the ability to utilize CO or syngas include: Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, and

Clostridium neopropanologen, Pseudomonas carboxidovorans, Rhodospirillum rubrum, Thermincola carboxydiphila, Thermincola potens, Thermoanaerobacter thermohydrosulfuricus, Ralstonia eutropha, Eurobacterium limosum. A CI

metabolizing bacterium also includes bacteria that can cleave methyl groups from organic compounds including choline (de Vries et al, 1990, FEMS Microbiol. Rev. 6:57-101) or the pesticide carbofuran (Topp et al, 1993, Appl. Environ. Microbiol. 59:3339-3349) and utilize them as sole sources of carbon.

CI metabolizing organisms include bacteria, yeast (not including Pichia pastoris), and Archaea. A CI metabolizing organism may be obligate or facultative. Examples of CI metabolizing organisms include: Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, Pseudomonas, Candida, Yarrowia, Hansenula, Pichia (not including Pichia pastoris), Torulopsis, and Rhodotorula. Additional examples of CI (carbon monoxide) metabolizing organisms include: Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium Woodii, and Clostridium neopropanologen.

A variety of CI substrates may be used by the CI metabolizing organisms. It is understood to one of skill in the art that selection of a CI substrate may be determined by which host organism is selected. For example, obligate

methanotrophic bacteria are limited to the use of methane as a carbon source, whereas some methylotrophic bacteria may use a variety of CI compounds, such as methane, methanol, methylated amines, halomethanes, and methylated compounds containing sulfur (reviewed in Hanson and Hanson, 1996, Microbiological Rev. 60:439-471). Non- limiting examples of CI substrates include: methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines

(methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, or methyl halogens (bromomethane, chloromethane, iodomethane, dichloromethane, etc.). Some facultative methanotrophs and facultative methylotrophs can also grow on multi-carbon compounds. A CI metabolizing organism may also be adapted or genetically modified to use a different CI substrate in addition to, or instead of its usual CI substrate.

Alternatively, a CI metabolizing organism may be adapted or genetically modified to use a multi-carbon substrate in addition to its usual CI substrate. A selected CI metabolizing organism may also undergo strain adaptation under selective conditions to identify variants with improved properties for production. Improved properties may include increased growth rate, yield of desired products (e.g., propylene), and tolerance of likely process contaminants. In a particular embodiment, a high growth variant CI metabolizing organism, which is an organism capable of growth on a CI substrate as the sole carbon and energy source and which possesses an exponential phase growth rate that is faster (i.e., shorter doubling time) than its parent, reference, or wild-type organism, is selected ((see, e.g., U.S. 6,689,601).

Each of the organisms of this disclosure may be grown as an isolated culture, with a heterologous organism that may aid in growth, or one or more of CI metabolizing bacteria may be combined to generate a mixed culture.

Microbial Organisms

A variety of microbial host organisms can be transformed or genetically engineered to include a novel metabolic pathway and associated enzymes as described herein to produce propylene from a carbon substrate. Exemplary bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobio spirillum succiniciproducens,

Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Cory neb acterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi species include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,

Aspergillus niger, Rhizopus arrhizus, Rhizobus oryzae, and Yarrowia lipolytica. It is understood that any suitable microbial host organism can be used to introduce suitable genetic modifications (e.g., nucleic acid encoding 4-OD) to produce propylene.

Non-naturally Occurring Organisms

Non-naturally occurring organisms as described herein can be produced by introducing expressible nucleic acid(s) encoding one or more enzymes involved in a desired biosynthetic pathway (e.g., propylene synthesis). Depending on the host organism selected for biosynthesis, nucleic acid(s) for one, some, or all of a particular biosynthetic pathway can be expressed. For example, if a selected host is deficient in one or more enzymes for a desired biosynthetic pathway (e.g., propylene synthesis), then expressible nucleic acid(s) for the deficient enzyme(s) are introduced into the host for subsequent exogenous expression. Alternatively, if a selected host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) to achieve the desired biosynthesis. Thus, non-naturally occurring organisms of the invention can be produced by introduced exogenous enzyme activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme activities that, together with one or more endogenous enzymes, produces the desired product, such as propylene. In some embodiments, non-naturally occurring organisms as described herein can also include other genetic modifications that facilitate or optimize a desired biosynthetic pathway or that confer other useful functions onto the host. For example, if a selected host exhibits endogenous expression of an enzyme that inhibits propylene biosynthetic pathway, then the host may be genetically modified so that it does not produce a functional enzyme or a substantial amount of a functional enzyme.

In certain embodiments, the present disclosure provides a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI

metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the non-naturally occurring CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non- naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO, wherein the non- naturally occurring CI metabolizing bacterium or organism converts a CI substrate to propylene.

In certain embodiments, the present disclosure provides a non-naturally occurring CI metabolizing organism that is not Pichia pastoris and is capable of converting a CI substrate into propylene. In certain embodiments, the non-naturally occurring C 1 metabolizing organism is not Pichia pastoris and is a C 1 metabolizing bacterium capable of converting a CI substrate into propylene.

In certain embodiments, the non-naturally occurring CI metabolizing organism is an obligate CI metabolizing organism capable of converting a CI substrate into propylene. In certain embodiments, the obligate CI metabolizing organism is a CI metabolizing bacterium, such as a methanotrophic or methylotrophic bacterium capable of converting a CI substrate into propylene. In further embodiments, a CI

metabolizing bacterium may be a syngas utilizing bacterium that naturally possesses the ability to use syngas and convert a CI substrate in the syngas into propylene. In still further embodiments, a CI metabolizing bacterium is a CO utilizing bacterium that naturally possesses the ability to use CO and convert the CO into propylene.

In certain embodiments, any of the non-naturally occurring CI metabolizing organisms as described herein includes an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid. The exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid is expressed in a sufficient amount to produce propylene.

In further embodiments, the exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid encodes 4-oxalocrotonate decarboxylase (4- OD). 4-OD is an enzyme that catalyzes the decarboxylation of 4-oxalocrotonate to 2- oxopent-4-enoate and C0 ₂ . 4-OD is involved in the meta-cleavage pathway for the degradation of phenols, modified phenols, and catechols. As disclosed herein, an exogenous nucleic acid encoding 4-OD is used to genetically engineer a novel propylene biosynthesis pathway in a non-naturally occurring organisms; 4-OD is used to catalyze decarboxylation of crotonic acid to propylene and C0 ₂ (see Figure 1).

Sources of 4-OD encoding nucleic acid molecules may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing decarboxylation of crotonic acid to propylene and C0 ₂ . Exemplary amino acid sequences of 4-OD are shown in Figures 2A-D.

Non-naturally occurring CI metabolizing organisms that have an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid {e.g. , 4-OD) may also include one or more exogenous nucleic acids to confer a propylene biosynthetic pathway onto the C 1 metabolizing organism. For example, depending on which CI metabolizing host organism is selected, one or more exogenous nucleic acids may need to be introduced into the CI metabolizing organism along with 4-OD in order to provide a non-naturally occurring C 1 metabolizing organism with the ability to produce crotonic acid, the substrate for propylene conversion by 4-OD. In certain embodiments, a non-naturally occurring CI metabolizing organism that has an exogenous nucleic acid encoding an enzyme capable of decarboxylating crotonic acid (e.g., 4-OD) may further include an exogenous nucleic acid encoding crotonase and/or an exogenous nucleic acid encoding a crotonyl-CoA thioesterase. An exogenous nucleic acid encoding crotonase and an exogenous nucleic acid encoding crotonyl-CoA thioesterase are expressed in a sufficient amount to produce propylene. In a specific example, a type II methanotrophic bacterium, which possesses an endogenous PHB synthesis pathway and produces 3-hydyroxybutyryl-CoA, an intermediate in the PHB synthesis pathway, may comprise exogenous nucleic acids encoding a crotonase and/or a crotonyl-CoA thioesterase in addition to 4-OD (see Figure 1). Crotonase and crotonyl-CoA thioesterase are provided to catalyze the dehydration of 3- hydroxybutyryl-CoA to crotonyl-CoA and conversion of crotonyl-CoA to crotonic acid, respectively. It is understood that any combination of one or more enzymes that can be used to engineer a propylene biosynthesis pathway can be included in a non-naturally occurring C 1 metabolizing organism of the invention.

In certain embodiments, the present disclosure provides a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI

metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the non-naturally occurring CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non- naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO, wherein the non- naturally occurring C 1 metabolizing bacterium or organism comprises an exogenous nucleic acid encoding a crotonyl-CoA thioesterase.

In certain embodiments, the present disclosure provides a non-naturally occurring C 1 metabolizing organism that is not Pichia pastoris and includes an exogenous nucleic acid molecule encoding a crotonyl Co A thioesterase. In further embodiments, a non-naturally occurring CI metabolizing organism that is not Pichia pastoris, may be an obligate CI metabolizing organism containing an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In still further

embodiments, the CI metabolizing organism may be a CI metabolizing bacterium or an obligate CI metabolizing bacterium containing an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In some embodiments, a CI metabolizing bacterium is a methanotrophic or methylotrophic bacterium containing an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In certain embodiments, a CI metabolizing bacterium may be a syngas utilizing bacterium that naturally possesses the ability to use syngas and contains an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase. In some embodiments, a CI metabolizing bacterium may be a CO utilizing bacterium that naturally possesses the ability to use CO and contains an exogenous nucleic acid molecule encoding a crotonyl CoA thioesterase.

Crotonyl-CoA thioesterase refers to an enzyme that catalyzes the conversion of crotonyl-CoA to crotonic acid. Sources of crotonyl-CoA thioesterase encoding nucleic acids may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing the conversion of crotonyl-CoA to crotonic acid. Exemplary amino acid sequences for crotonyl-CoA thioesterase are shown in Figure 3. Crotonic acid is an intermediate in the engineered propylene biosynthetic pathway disclosed herein and is a useful product in itself for the preparation of polyvinyl acetate copolymers, a synthetic butyl rubber softener, a variety of resins, fungicides, pharmaceutical intermediates, plasticizers, cosmetic polymers for hair care, and adhesives. Reduction of crotonic acid yields crotonaldehyde. Key products made from crotonaldehyde are sorbic acid, potassium sorbate (a preservative), and trimethylhydroquinone (an intermediate used in Vitamin E manufacture).

In certain embodiments, the present disclosure provides a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI

metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the non-naturally occurring CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non- naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO, wherein any of the aforementioned non-naturally occurring C 1 metabolizing bacterium or organism includes an exogenous nucleic acid encoding a crotonase.

In certain embodiments, the present disclosure provides a non-naturally occurring C 1 metabolizing organism that is not Pichia pastoris and includes an exogenous nucleic acid molecule encoding a crotonase. In certain embodiments, the non-naturally occurring C 1 metabolizing organism that is not Pichia pastoris is a C 1 metabolizing bacterium containing an exogenous nucleic acid molecule encoding a crotonase, such as a methanotrophic or methylotrophic bacterium. In some

embodiments, a CI metabolizing bacterium is a syngas utilizing bacterium that naturally possesses the ability to use syngas and contains an exogenous nucleic acid molecule encoding a crotonase. In some embodiments, a CI metabolizing bacterium is a CO utilizing bacterium that naturally possesses the ability to use CO and contains an exogenous nucleic acid molecule encoding a crotonase. In further embodiments, a non- naturally occurring CI metabolizing organism is an obligate CI metabolizing organism containing an exogenous nucleic acid molecule encoding a crotonase, such as an obligate CI metabolizing bacterium.

Crotonase, also known as 3-hydroxybutyryl-CoA dehydratase, is an enzyme involved in the butyrate/butanol-producing pathway. Crotonase catalyzes the dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA. 3-hydroxybutyryl-CoA can be an R or S stereoisomer. Enzymes that produce and convert 3-hydroxybutyryl-CoA have defined stereospecificity preferences. Sources of crotonase encoding nucleic acids may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA.

Exemplary amino acid sequences for crotonase are shown in Figures 4A-E. Crotonyl- CoA is a useful intermediate that can be a substrate for an engineered propylene biosynthetic pathway (via conversion to crotonic acid by crotonyl-CoA thioesterase, which is then converted to propylene by 4-OD) or an engineered butanol/butyraldehyde biosynthetic pathway (via conversion to butyryl-CoA by butyryl-CoA dehydrogenase, which is then converted to butanol or butyraldehyde by bi-functional aldehyde dehydrogenase) (see Figure 1).

In certain embodiments, the invention provides for a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI

metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the non-naturally occurring CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non- naturally occurring CO utilizing bacterium, wherein the non-naturally occurring CO utilizing bacterium naturally possesses the ability to utilize CO; wherein the non- naturally occurring CI metabolizing bacterium or organism converts a CI substrate to propylene, and further wherein the non-naturally occurring C 1 metabolizing bacterium or organism does not have a functional PHB synthase or a substantial amount of a functional PHB synthase.

In certain embodiments, a non-naturally occurring CI metabolizing organism according to any of the embodiments disclosed herein is a CI metabolizing bacterium that does not have a functional PHB synthase or a substantial amount of functional PHB synthase.

PHB synthase is a polyhydroxybutyrate synthesis pathway enzyme that converts hydroxybutyryl-CoA monomers into polyhydroxybutyrate (PHB) (see Figure 1). Many prokaryotes synthesize PHB as a carbon and energy storage material. Some yeast and other eukaryotic cells may contain small amounts of low molecular mass PHBs. Two PHB synthesis pathways are known in bacteria. In one pathway, PHB is synthesized from acetyl-CoA as a result of sequential action of three enzymes: β- ketothiolase, acetoacetyl-CoA reductase, and PHB synthase (e.g. , Methylobacterium extorquens, Methylosinus trichosporium OB3b, Methylocystis and Methylosinus species). PHB synthesis can also be synthesized by: β-ketothiolase, acetocetyl-CoA reductase, crotonyl-CoA hydratases (crotonases), and PHB synthase (e.g.,

Methylobacterium rhodesianum) (see, e.g., Mothes et al, 1994, Arch. Microbiol.

161 :277-280; Mothes et al, 1995, Can. J. Microbiol. 41 :68-72). Generally, Type II methanotrophs accumulate PHB, whereas type I methanotrophs do not. As used herein, "not having a functional PHB synthase" means that its gene expression or protein activity has been reduced to undetectable levels. Reducing gene expression or protein activity of PHB synthase may be accomplished by deletion of some or all of the gene's coding sequence, introduction of one or more nucleotides into the gene's open reading frame resulting in translation of a nonsense or non- functional protein product, expressing interfering RNA or antisense sequences that target the gene, or other methods known in the art. As used herein, "not having a substantial amount of a functional PHB synthase" means that an organism has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less gene expression or protein activity of PHB synthase as compared to a wild type organism that has a

polyhydroxybutyrate synthesis pathway.

In a specific embodiment, PHB synthase is encoded by phaC or phbC. Exemplary PHB synthase amino acid sequences are provided in Table 1. In another specific embodiment, a non-naturally occurring CI metabolizing organism that is capable of converting a C 1 substrate to propylene as disclosed herein does not produce a substantial amount of polyhydroxybutryate. As used herein, "not producing a substantial amount of polyhydroxybutyrate" means that an organism produces at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less polyhydroxybutyrate as compared to a wildtype organism that has a

polyhydroxybutyrate synthesis pathway. By knocking out PHB function in CI metabolizing organisms with an endogenous PHB synthesis pathway through genetic modification (e.g., allelic exchange of phaC or phbC with non- functional gene), PHB production may be inhibited, allowing more 3-hydroxybutyryl-CoA to be funneled into conversion to crotonyl-CoA by crotonase (see Figure 1). Alternatively, a CI metabolizing organism, which is not naturally capable of PHB synthesis, may be genetically modified to possess a portion of the PHB synthesis pathway (e.g. , β- ketothiolase and acetoacetyl-CoA reducatase), while excluding PHB synthase functionality. Increased amounts of crotonyl-CoA, which is converted to crotonic acid and then to propylene via crotonyl-CoA thioesterase and 4-OD, respectively, may result in increased propylene yields.

Table 1 : Exemplary PHB synthase sequences

Genbank Gene Name Amino Acid Sequence (SEQ ID NO:#)

Accession #

[Legionella AYYSWFAQLLQSPGSMLRLAYYPLLHANDYLSNLFKYDKPRDGKDVRFHTENWSYYPWRL pneumophila WAEQFLQFEDWCLQASSKVPGIPLHVKRTVTFSTRQILDALSPSNFVLTNPDLLQETIRS NGQ str. Corby] NLIRGTELAFQDFVEKITGSPPAGVENFIPGKQVAVTKGKVVYSNHLIELIQYTPQTEKV YKEPI

LILPAWIMKYYILDLLPENSLVNWLVRQGHTVFIVSWRNPTKEDRNLGLDDYYKLGAMDA IN

AVSNAIPHTKIHLMGYCLGGTLALLTAAAMAHDHDNRLKTLSLLAAQGDFIDAGELL LFITKS

EVSFLKSMMWEQGYLDTKQMSGTFQMLRSYDLIWSKMVQDYMHGTQRGMIPLLAWNA

DATRMPYKMHSEYLEKLFLNNDFAEGRFILDGKPVVGENIRIPAFVVSTEKDHVAPW KSVYK

THLLINSDITFVLTNGGHNAGIVSEPGHEGRYYRIRERKMDSTYLDPTNWLKKAELR EGSWW

IAWHDWLVNHSSQKQVSAPKLDKKLPNAPGKYVLQK (SEQ ID NO:58)

gi 1326404096 poly(3- MPGFATFDRLSRAMFARISQGVSPLAVADAWTDWALHLELALGKQEALAMRAATFLLRLG hydroxyalkan FWLPRAAIGEPENPPLRPPEGDRRFADEGWSAYPFNAIVQGFLVADDWWREATRQVPGLV oate) RRHEAEVAFMARQILDIAAPTNIPWLNPEIIRRTLQEGGFNLLRGWSNWIEDAERLLAGQ GP polymerase YGAEAFPVGEVVATTAGKVVYRNELMELIQYAPATAKVTAEPVLIVPAWIMKYYILDLTP ETS [Acidiphilium LVRYLVTNGHTVFIISWINPDRHDRNVGLDDYRRHGVMAALDAVSRIVPDRAIHACGYCL G multivorum GTILAIAAATMARDHDDRLASLTLLAAQTDFADAGDLMLFLDERQFLLLEDLMWDQGYLD T AIU301] RQMAGAFQALRSNELVWSRLIRNYMLGERDRMTPLSAWNSDQTRMPARMHSEYLRGLFL

ENRLSAGRFAVEGRVIALRDIRVPLFAVATTRDHIAPWRSVYKIALFADTDITFVLASGG HNV

GVVNPPNQSIGTFQILTRRHGERYVDPDTWATLAPERQGSWWPAWRDWIGGVGTGCA A

DPPPMAAPSRGLPVLGDAPGAYVHER (SEQ ID NO:59)

gi 1330826932 poly-beta- MPDTFSAAALADQLDTQFHAALARRFFSLSPAAGMLAASDWALHLAVSPGKCMALARLAL hydroxybutyr RQSEELAGYARERMTAGADPQLRHGVQPPAQDRRFAAPEWQQWPFNYMHQSFLLTQQ ate WWAAATHGVKGVEKHHENVVAFAARQLLDVFSPGNQLATNPVVLQRTLQQGGANLLRG polymerase ALNAADDLQRLAAGKPPAGTEDFVVGRDVAVTPGKVVLRNRLVELIQYTPTTEAVHPEPV LI domain- VPAWIMKYYILDLSPHNSLIRYLVDQGHTVFCLSWKNPGYEDRDLGLDDYLKLGFHAALD AV containing NAIVPKRKVHATGYCLGGTLLAIAAAAMARDGDARLASLSLFAAQTDFSEPGELGLFIDE SQV protein NLLEAQMTQTGYLKASQMAGAFQMLRSYDLLWSRLVNEYLLGERTPMNDLMAWNADAT [Alicycliphilus RMPARMHAQYLRRLLLDDDLSRGRYPVGGKPVSLSDITLPIFMVGTLTDHVAPWRSVHKL H denitrificans HLTTTEITFALTSGGHNAGIVNPPGNPRRHYQLRTRPAGGNYMAPDDWLAAAPAAQGSW K601] WPAWQEWLQARSGKPVAPPHMGARGYKAGADAPGHYVLEK (SEQ ID NO:60) gi 1222110509 poly-beta- MSNVATHGKADLAQPEACRPDTLDTLANAWRARSTGGLSPAAGLLAWYDWALHLSLSPG hydroxybutyr KQRSLIEKGLHKQQRLARYALRVASAHDCPTCIEPLEQDRRFAAPAWQQWPFNVIHQGFL L ate QQQWWHNATTGVRGVSRHHENMVTFAGRQWLDMWSPSNFIWTNPEVLHAITQSGGA polymerase NLWRGAMNFLEDARRLALDDAPAGVEGFEVGKDVAVTPGKVVFRNHLIELIQYSPTTPDV H domain- AEPVLIVPSWIMKYYILDLSPHNSMVKYLVDQGHTVFILSWKNPTAADRDLGLEDYRWLG V containing MDALDAVTAIVPERKVQAVGYCLGGTLLAIAAAAMARDGDERLQSLTLLASETDFRESGE IAL protein FIDDSQLAWLEAGMWDKGYLDGKQMAGAFQMLNSRDLIWSRRVREYLLGERQTFNDLM [Acidovorax AWNADVTRMPYRMHSEYLRRLYLDNDLAEGRYRVGGRPVALADIEVPMFIVGTVRDHVAP ebreus TPSY] WPSVYKMHLLSDAELTFVLTSGGHNAGVVSEPGHPRRSFQIATRAAGDRYIDPQLWRAET P

MNEGSWWPAWQQWLAQRSAGRVAPPAMGGTQAPLGDAPGTYVAMR (SEQ ID

NO:61)

gi 1 39937303 phaC gene MMLQTSPPAPSAASQQIQSPARHHGPSDSDRTLHATLAPLTGGLSPTALTLAYADWLSHL F product WAPTQRMDLVNDALRRGTQLAEASIGQTAPWSLIAPQPQDRRFSAPEWREPPFNLMAQG

[Rhodopseud FLLAEQWWHDATTNIRGVSEQNEKIVEFATRQMLDVWAPSNFALTNPEVLRRTVSTEGRN L omonas ADGFRNWWEDLLELMAHEPKHDFVVGKDVAVTPGKVVYSNKLIELIQYAPATETVRPEPI LI palustris VPAWIMKYYILDLSPHNSLVKYLTEQGYTVFMISWRNPTAADRDVSLEDYRRLGVMAALD TI

CGA009] GAILPDRPVHAVGYCLGGTMLSIAAAAMGRDGDSRLKSITLFAAQTDFTEAGELTLFINE SQV

AFLEDMMWNRGVLDTTQMSGAFQILRSNDLIWSRLVHEYLMGVRSEPNDLMAWNADAT

RMPYRMHSEYLRKLFLDNDLAEGRYVVDGRPIALSDIHTPIFVVGTQRDHVAPWRST FKIHLL

ADADVTFCLTGGGHNAGIVSPPSPKAHGYQVMTKEADGPYVGPDDWLKQAPHAEGSW W

TEWVHWLGTRSGEPVAPPRIGLPDVDPGALPNAPGSYVLOJ (SEQ ID NO:62) gi 1 172063316 poly-beta- MKASVTRAPRGNESIRPSGAVEPESSMTQCPQAGTTPAEQWNRAAHANVAAMTFGLSPV hydroxybutyr SLALAMLDWGAHLAVSPGKCFDLAMQACVAAVAPAADQCEAEAGEADPTGLAVHAGQA ate DPRFAAPAWAGWPFHVYRDSFLSIQRWWRDATHGVPGVERHHEELVGFAARQWLDACS polymerase PGNFLATNPVVLDATMCSGGANLAAGALNWLEDAKALLERAGGTHAHDARTYLPGRDVAI domain- TPGRVVWRNALCELLQYEPTTARVAREPILIVPSWIMKYYILDLQPHNSLIRFLVDAGYT VFAV containing SWRNPGAEARDLGLDDYLRDGCMAALDAARSVCGGAVHTVGYCLGGTLLAIVAAALARDG protein RQHEALRSVTLLAAQTDFSEPGELGLFIDASELSALDALMWRQGYLDGAQMSAAFQLLNA R Genbank Gene Name Amino Acid Sequence (SEQ ID NO:#)

Accession #

[Burkholderia DLIWSRMMSEYLLGTRTKPNDLMSWNADTTRMPYRMHSEYLTRLFLDNDLAVGRYCVDG ambifaria RPVALSDIDVPTFVVGTERDHVSPWGSVYKLHLLTHHALTFVLTSGGHNAGIVSEPGHPG RH

MC40-6] YRRATREPGAPYRSRHDFVRGTTAVDGSWWTCWRDWLHERSSGDVPARTPAAGFDAAP

GTYVLET (SEQ ID NO:63)

gi 1 1730539 PHAC_METEX MGTERTNPAAPDFETIARNANQLAEVFRQSAAASLKPFEPAGQGALLPGANLQGASEIDE M

RecName: TRTLTRVAETWLKDPEKALQAQTKLGQSFAALWASTLTRMQGAVTEPVVQPPPTDKRFAH

Full=Poly(3- ADWSANPVFDLIKQSYLLLGRWAEEMVETAEGIDEHTRHKAEFYLRQLLSAYSPSNFVMT NP hydroxyalkan ELLRQTLEEGGANLMRGMKMLQEDLEAGGGQLRVRQTDLSAFTFGKDVAVTPGEVIFRND oate) LMELIQYAPTTETVLKRPLLIVPPWINKFYILDLNPQKSLIGWMVSQGITVFVISWVNPD ERH polymerase; RDKDFESYMREGIETAIDMIGVATGETDVAAAGYCVGGTLLAVTLAYQAATGNRRIKSAT FL

Short=PHA TTQVDFTHAGDLKVFADEGQIKAIEERMAEHGYLEGARMANAFNMLRPNDLIWSYVVNNY polymerase; VRGKAPAAFDLLYWNADATRMPAANHSFYLRNCYLNNTLAKGQMVLGNVRLDLKKVKVP

AltName: VFNLATREDHIAPALSVFEGSAKFGGKVDYVLAGSGHIAGVVAPPGPKAKYGFRTGGPAR GR

Full=PHA FE DWVAAATEH PGSWWPYWYKWLE EQAPE RVPARI PGTG ALPSLAPAPGTYVRM KA synthase; (SEQ ID NO:64)

AltName:

Full=Polyhydr

oxyalkanoic

acid synthase

gi 1 16125629 poly-beta- MVETLSANLARAAVTAQGAIAEAALRQADRPAALTPDPFHVAPALNEVMTRLAAQPDRLM hydroxybutyr RAQADLFGQYMELWQTAARRAAGEDVAPVVAPAAGDKRFNDPDWASNPMFDLMKQSY ate LLSSNWLNGLIAEVDGVDPATKRRVEFFTKMLTDAFSPSNFLISNPAALREVVQTQGQSL VR polymerase GMENFAADLERGGGQLAISQTDLAKFKVGENVATAPGKVVYQNDILQLLQFDPTTDTVCE IP [Caulobacter LLIFPPWINKFYIMDLRPENSMIRWLTAQGFTVFVASWVNPDQTLAAKTFEDYMVEGIYD A crescentus AQQVMTQCGVDRVNTVGYCIGGTLLSVALAHMAARGDKRINSATFFAAQQDFAEAGDLLL CB15] FTNEEWLQSIEQQMDQAGGFLPSQSMADTFNALRGNDLIWSFFVSNYLMGKEPRPFDLLF

WNADQTRMPKALHLFYLRNFYKDNALTTGKLSLGGERLDLSKVKIPIYVQSSKDDHIAPY RSV

YRGARAFGGPVTFTMAGSGHIAGVINHPDARKYQHWTNSELPADVSEWIAGAHEHPG SW

WPHWAAWLKARSGDQVPARDPAKGKLKPLEDAPGSFVLVKSQP (SEQ ID NO:65) gi 1 170744156 unnamed MLATSPTQSSAAIPARPPALRLVPPVAAAGPVRDAAEPGDDLHDAGQAIDQAAHAAVARL T protein GGLSPAALANAWLDWSVHAAFSPGKQAELAAKAFRKGQRLQSFLWRNLLVGAQAEPCIEP product LPQDHRFDDPAWRTWPFCLYQQGFLLTQQWWHNATTGVRGVARAHEEIVAFTARQMLD

[Methylobact GVSPSNHPLTNPVVLNATLASGGANLVLGALNALEDARRGLAGLKPAGAERFAVGREVAV T erium sp. 4- EGEVVYRNDLIELIQYAPKTGAVRPEPVLIVPAWIMKYYILDLSPENSLVRHLVGQGFTV FMIS

46] WRNPGPADRDVSFDDYRRLGVMAALDAVSAIRPGRAVHAAGYCLGGTLLSIAAATMARDG

DARLASLTLFAAQVDFTEAGELTLFINESQISFLESLMRSEGVLDSKQMAGAFQLLRSND LIW

SRIVNSYLLGRREAVTDLMAWNADATRMPARMHAEYLRRLFLDNDLAEGRLRVEGRP VAL

TDIRAPIFAVGTEKDHVAPWRSVFKLTLMTDADVTFLLASGGHNAGIVSEPGHRGRH YRVHS

RAATDRYVDPDSWLDLARLEQGSWWPEWASWLAERSGPPEPPPPMGLPGAPTLGPAP G

RYVREA (SEQ ID NO:66)

gi 1 146340526 Poly-beta- MTDVPN NTN PQKTFDAEAFATN 1 ARAM ESSG KALAAYLKPRETG EVQDRPPTE LTEVVKSFT hydroxybutyr AVADYWLSDKDRASDIQTKLAKGYLDLWGSAARRLAGEEAPPAISPSPRDKRFADPEWKS N ate QFYDFVMQAYLLTTQWAQDLVHNAEGLDPHTRKKAEFYVNQITNALAPSNFVMTNPEVM polymerase RQTVASSGDNLVRGMQMLAEDIEAGKGTLKIRQSDPANLEVGVNMATTPGKVIFQNEMM [Bradyrhizobi QLIQYAPTTETVLRTPLLIVPPWINKFYILDLRPEKSFIKWCVDQGLTVFVISWVNPDKK LGTKT urn sp. ORS WEDYMKEGPLTAMDVIEKVTGEMKVHTMGYCVGGTMLATTLAWLADKRRQRVTSATFLA 278] AQVDFTHAGDLLVFVDETQISALERDMQASGVLEGSKMAMAFNMLRSNDLIWSYVVNNYL

KGQPPQAFDLLHWNSDATRMSAANHSYYLRNCYLENRLTTGTMVLDNTLLDLSKVKVPVY

NLATREDHIAPADSVLYGSQFFGGPVKYVLSGSGHIAGVVNPPSSGKYQYWTNDQIH DISLK

DWMKGAQEHKGSWWPDWREWLGQLDPEQVPARSVGSEAYPPIEDAPGSYVRVRA

(SEQ ID NO:67)

gi 1 126463677 unnamed MSDMKWNAEGAPAYGQALDRAARAAIAGMTRGLAPSVLATAALDWMMHLAAAPGKQA protein ELWEKAATASAALMQAGLQPHEAPVRDRRYASEAWNRQPFAALRDSFLLTEDWWQTATT product GLRGMDRAHEAALSFSVRQMLDVWSPSNNPFLNPEVLARTTETRGANLMQGAMNFAGD

[Rhodobacter MARLATGVPMDEGGFRIGETLAATPGKVVLRTHLMELIQYSPTTREVHPEPVLIVPAWIM KY sphaeroides YILDLSEQNSLVRWLVAQGFTVFMISWRNPESEDRDLGLIDYLDQGPRAALKAIQTITGA PKV

ATCC 17029] HAAGYCLGGTLLSIMAARMAHDHDERLASMTLFAAQVDFSEAGELALFISEAQVALLEDM Genbank Gene Name Amino Acid Sequence (SEQ ID NO:#)

Accession #

MWHQGYLDSDQMSGAFTLLRSNDLIWSRMIHEYMMGERPHPNDLMTWNADSTRMPYR MHSEYLRQLFLENRFAEGKFELEGHALSLTELRLPILAVGTETDHVAPWRSVFKIQRLTE TETT FVLTSGGHNAGIVSEPGHPRRHFRIATTGRDDPYRDADEWFAETAPVEGSWWPAWGAWL AERSTPKGKLPPMGNARGGYPALCEAPGTYILQR (SEQ ID NO:68)

gi 1359401847 putative MKGLKI M VE AQI PG VG DPLDG LAETM DRAAG AM 1 AQATFG LSPATLAQAVSDWM LH LAA poly-beta- SPGKQTQLAAKALRKMTRLGDYAMRSATDAQAARAIEPLPQDRRFADPAWASAPFNLVSQ hydroxyalkan AFLLNQQWWHAATTGITGVTAHHEEMVAFAVRQWLDTVAPTNFLATNPVLQQRILETGG oate synthase QCLADGLRNWMTDVEALMRGLPPAGTEAFQVGETLATAEGKVVYRNRLMELIQYAPTTEQ

[Novosphingo ARPEPILIVPAWIMKYYILDLSPENSLVQWLTAQGFTVFMISWHNPGSTDRDLEMADYLQ LG bium PMAALDAVAAITGGASVHAAGYCLGGTLLAIAAAAMARDGDDRLASLTLLAAOJEFCEPG E pentaromativ LGLFIDEGQLSLLENMMWGRGYLDSAQMGGAFQMLRSNDLVWSRVLTTYLMGEREPMN orans US6-1] DLMAWNADGTRMPYAMHSQYLRRLFLEDDLAEGRFQVNGRPIALSVLRWPMFVVGTERD

HVAPWRSVFKIHRLTGAPIDFVLTSGGHNAGIVSEPGHPGRSYRLLTREADGAALDPDAW LD

AAPRHEGSWWTAWGDWLAKLSGNAGTPPPMGATDKGYAPLADAPGHFVLER (SEQ ID

NO:69)

gi 1 581529 PHA synthase MLDHVHKKLKSTLDPIGWGPAVTSVAGRAVRNPQAVTAATAEYAGRLAKIPAAATRVFNA

[Rhodococcus NDPDAPMPVDPRDRRFSDTAWQENPAYFSLLQSYLATRAYVEELTEAGSGDPLQDGKARQ ruber] FANLMFDALAPSNFLWNPGVLTRAFETGGASLLRGARYAAHDILNRGGLPLKVDSDAFTV G

ENLAATPGKVVFRNDLIELIQYAPQTEQVHAVPILAAPPWINKYYILDLAPGRSLAEWAV QH

GRTVFMISYRNPDESMRHITMDDYYVDGIATALDVVEEITGSPKIEVLSICLGGAMA AMAAA

RAFAVGDKRVSAFTMLNTLLDYSQVGELGLLTDPATLDLVEFRMRQQGFLSGKEMAG SFD

MIRAKDLVFNYWVSRWMKGEKPAAFDILAWNEDSTSMPAEMHSHYLRSLYGRNELAE GLY

VLDGQPLNLHDIACDTYVVGAINDHIVPWTSSYQAVNLLGGDVRYVLTNGGHVAGAV NPP

GKRVWFKAVGAPDAESGTPLPADPQVWDEAATRYEHSWWEDWTAWSNKRAGELVAPP

AMGSTAHPPLEDAPGTYVFS (SEQ ID NO:70)

gi 1227823933 poly-beta- MKTGDRPVLAADRARQAGSARAIAAQSALPCENDGADGEAFRAIDRMREALSATATGGLS hydroxybutyr PAALTLAFFDWSIHLASAPGKRMELAHMAAQNWGLLLTYMAAAATRPDAPPCIEALPGDN ate RFRAEGWQKQPYTVWAQAFLLGQQWWHNVTRNVPGMTPHHEDVVSFTARQWLDVFS polymerase PSNIPFANPEVIHKAMETGGANFTQGFRNWLEDVGRLATKQRPVGTEAFRVGHDTAATPG domain KVVYRNHLIELIQYAPATEEVLAEPILIVPAWIMKYYILDLSPHNSLIRYLVAQGHTVFC ISWRN protein PSAKDRDLTLDDYRRLGILAALDAVSAIVPERKIHATGYCLGGTLLAIAAAAMARTEDQR LAS

[Sinorhizobiu VTLFAAQTDFSEPGELALFIDHSQLHFLDSLMWHSGCLSADQMAGAFQLLRTNDLVWSRL V m fredii HDYLIGKRTPMTDLMAWNADPTHMPYRMHAEYLQRLYLDNELAAGRFIVDGRPAHLQNIR

NGR234] VPMFVVGTERDHVAPWHSVYKIHYLTDTDVTFVLTSGGHNAGIVSEPDHPGRGFRIALTR ES

DSSVSADEWVAAATSKDGSWWPDWVEWLAGHSAPKRVTPPVIGAPERGYPPIDDAPGTY

VHQR (SEQ ID NO:71)

gi 1332526305 poly-beta- MHNPNVAAAVLADPALDPALAAARRLDSHFHAAVAPAFSGLSPISLALAWQDWALHLATQ hydroxybutyr PATALALVARAQQSWLQAWGEMLGQAEPGNGDARFAAPAWRQWPWAPVVHGWHAT ate ERWWQDATDLRGVDPHHREVVRFFARQWLDMLAPSNAGLANPEVLQRTAERGGANLVD polymerase- GTLHALDGWRRQHGLEPLRTPERRYEPGVDVAVTPGQVVWRNHLVELIQYLPLTASVQAE P like protein VFIVPSWIMKYYILDLSPHNSFVKWLVEQGHTVFILSWRNPDESDALLAMQDYLELGIFD PLA [Rubrivivax QIARMIPGRRVHACGYCLGGTLLSLAAAALARPGRIARAELLPELASVSLLAAETDFTEP GEM benzoatilyticu GVLIDESQVTLLEDMMAERGFLTGAQMAGSFAYLHSRDQVWSRRLREFWLGEPDSPNDL s JA2] MAWNADLTRMPAAMHSEYLRRCYLRNEIAEGRFPVEGRAVSLSDIAAPMFVVGTEKDHVS

PWKSVYKIHRLADTTITFVLTSGGHNAGIVSEPGHANRRYRMATREVDGAWVDPEAWAEQ

APRHEGSWWTAWHEWLLAQGSGETAKARTPAKADVVCAAPGTYVYQCWRD (SEQ ID

NO:72)

gi 1 154253888 unnamed MTKNKKGNTSSTIVPALDMQAHMAWAQAWSSISPESSLLAWTDWASHLANSPGKQSELL protein AFAGSLSEQWMSLLKKSLVSPDQEVASPEPSPTNDRRFNDPAWDQWPYNLFRSSFLIQSK W product WEQATQGVWGVDPQHERLLAFGAKQWLEMVSPTNSALFNPVVLKKTIEEQGANLARGM

[Parvibaculum SNFLDDLRRQLSGEPPAGTENFVVGRDVAVTEGKVVLRNQLIELIQYTPTTEKVHPEPIL IIPA lavamentivora WIMKYYVLDLSPHNSLIRYLVAQGHTVFCISWKNPDAGDRDLGMDEYLEFGLRAALDAVT SI ns DS-1] VPEQGIHAAGYCLGGTLLAIGASAMARDGDTRLVSVSLLAAQTDFSEPGELSLFINESQV ALL

EASMAQTGYLTSDQMSGAFQLLRSYDLIWSRMIDEYLLGDRRPMTDLMAWNADGTRLPA

KMHSQYLRRLYLNNDLSAGRYPVTGRPVSVGDIAVPVFCVGTASDHIAPWRSVYKLH LLSSA

ELTFVLTTGGHNGGIVSEPGRGKRQYQIHTRAAGDGYMAPDEWQATAQTHLDSWWPA W

SAWLRERSGEGVAPPLMGAESRGYPTICDAPGKYVRS (SEQ ID NO:73) Genbank Gene Name Amino Acid Sequence (SEQ ID NO:#)

Accession #

gi 1 53716799 phbC gene MDTRHAPESGAPDAPLPAHPPASYAPESPYRIFDLAKEASVAKLTSGLSPASLQLALADW LIH product LAAAPGKRAELATLALRHAALLGQYLLEAATGRTPAAPAQPSPGDRRFRAGAWQLEPYRF W [Burkholderia HQSFLLAEQWWRAATRDVPGVSPHHEDVVAFSARQMLDTFAPANYVATNPEIAQRTALT mallei ATCC GGANLAQGVWNYLDDVRRLITKQPPAGAEQFELGRNLATTPGRVVFRNHLIELLQYSPTT PD 23344] VYAQPVLIVPAWIMKYYILDLSAHNSLIRYLVGEGHTVFCISWRNVDASDRDLSLDDYRK LGV

MDALDTIGAIVPGEKIHATGYCLGGTLLSIAAAAMANTGDDRLASITLLAAQTDFAEPGE LQL

FIDDSEIHFLESMMWERGYLGAHQMAGSFQLLMSNDLIWSRVIHDYLLGERTPMIDL MAW

NADSTRMPYRMHSEYLRHLFLDNDLATNRYVIDGQTVSVHNIRAPFFVVGTEHDHIA PWRS

VYKIHYLSGSDVTFVLTAGGHNAGIVSEPGHAKRHYRMKMTAAAAPSISPDEWLAGA TDFE

GSWWPAWHAWLARHSSPQRVAPPPLGKPGAHTLGDAPGTYVFQK (SEQ ID NO:74) gi 1365891729 putative MSDAKSAAEDANSVAREFVREDHELDKAFSAVLARLTGGISPAALSMAYLDWACHLAAAP Q poly-beta- RQLDIARDAWQGARQLAERSLHFADSNRVPWDLIKPQANDHRFSKPQWGMQPFNLFAQ hydroxybutyr AFLLGEDWWHKATTNIRGVDPANEAVVDFSLRQLLDMFAPSNFAATNPEVVEKIFQSGGE ate NLVFGWQNWLSDLMQVLQPGQASRSAEFVPGETVATAPGKVVFRNQLIELIQYAPTTAEV polymerase RPEPILIVPAWIMKYYILDLSQHNSLVRYLTDQGFTVFMISWRNPDAKDRDIAFDDYRSM GV (Poly(3- MAALSEIGKIVPGAQIHAAGYCLGGTLLSITAAAMAREGDTRLKTITLFAAQTDFTEAGE LTLFI hydroxybutyr NESQVAFLEDMMWERGYLDTTQMAGAFQLLRSNDLIWSRVSRDYLLGERAHPSDLMAW ate) NADATRLPYRMHSEYLRKLFLNNDLAEGRYRVEGKPVSLSDIHTPMFVVGTLRDHVAPWK S polymerase)) TYKIHYEVDADVTFVLASGGHNAGIVAPPHEQGHSHQVRTKAADAPYLGPDEWQSTSPRI E

PHB/PHA GSWWPTWLDWLAQRSGPLDAPPRLGTEGSHELGNAPGEYVHS (SEQ ID NO:75) polymerase))

PHB/PHA

synthase)(Pol

y(3- hydroxyalkan

oate)

polymerase))

Polyhydroxyal

kanoic acid

synthase)

[Bradyrhizobi

urn sp. STM

3809]

gi 1338984011 Poly(R)- MAEQQNPRTEAPNLPDPAAFSRTMADIAARSQRIVAEWLQRQHEADVAIDPLNIGSAFME hydroxyalkan MMARLMANPASLIEAQIGFWQDYVTLWQHSTRRIMGIETQPVVPPDPRDKRFQHEEWKE oic acid NEIFDFIRQSYLLSARFVQDVVRRVDGLDPKTAQKVDFYARQFVDAMSPSNFALTNPQVL RK synthase, TAETGGENLLRGLSNLLRDLEAGRGQLHIRMTDAEAFSVGGNIAVTPGKVVYRNELMELI QY class 1 APATTTAHKTPLVIFPPWINKFYILDLRPKNSFIRWAVEQGHTVFVASWVNPDERLAEKD FA

[Acidiphilium DYMKLGVFAALDAIEQATGERQVNAIGYCLGGTLLAATLAVMARRRDSRIKSATFLVTLT DF sp. PM] ADVGELGVFIDEEQLAALEDRMNRRGYLEGSAMATTFNMLRSNDLIWSFVVNNYLLGNET F

PFDLLYWNSDSTRMPAAMHSFYLRNMYQKNLLSQADAITLDGTPVDLRRIKVPVYFLSCR ED

HIAPWASTYRATQLMAGPKRFVLAASGHIAGVINPPGSGKYNHFVNATLPANAEDWF AGA

TEVAGSWWPDWQRWI AAQG RG EVPARTPG DGALPALADAPG DYVKVRSA (SEQ ID

NO:76)

In certain embodiments, the present disclosure provides a non-naturally occurring CI metabolizing bacterium; a non-naturally occurring obligate CI

metabolizing organism; a non-naturally occurring CI metabolizing organism, wherein the non-naturally occurring CI metabolizing organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non- naturally occurring CO utilizing bacterium, wherein the non-naturally CO utilizing bacterium naturally possesses the ability to utilize CO; wherein the non-naturally occurring C 1 metabolizing bacterium or organism converts a C 1 substrate to propylene, and further wherein the non-naturally occurring CI metabolizing bacterium or organism has a functional β-ketothiolase and/or a functional acetoacetyl coenzyme A reductase, or has a functional β-ketothiolase and/or a functional acetoacetyl coenzyme A reductase activity (i.e., has enzymes that perform the function of β-ketothiolase and acetoacetyl coenzyme A reductase).

In certain embodiments, a non-naturally occurring CI metabolizing organism according to any of the embodiments disclosed herein is a CI metabolizing bacterium that has a functional β-ketothiolase activity and a functional acetoacetyl coenzyme A reductase activity.

In certain embodiments a non-naturally occurring CI metabolizing organism according to any of the embodiments disclosed herein is a CI metabolizing bacterium that does not produce a substantial amount of polyhydroxybutyrate.

β-ketothiolase and acetoacetyl coenzyme A reductase are enzymes in a PHB synthesis pathway that catalyze the condensation of two acetyl-CoA molecules to acetoacetyl-CoA and reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA, respectively (see Figure 1). β-ketothiolase is an enzyme that initiates biosynthesis of PHB in most bacteria. A C 1 metabolizing organism may naturally possess β- ketothiolase and acetoacetyl coenzyme A reductase as part of an endogenous PHB synthesis pathway. Alternatively, a C 1 metabolizing organism may be genetically modified with exogenous nucleic acids encoding β-ketothiolase and acetoacetyl coenzyme A reductase. These enzymes allow a CI metabolizing organism to produce 3-hydroxybutyryl-CoA, a precursor of propylene in an engineered biosynthetic pathway via crotonyl-CoA and crotonic acid as disclosed herein (i.e., 3-hydroxybutyrl-CoA is substrate of crotonase) (see Figure 1). Exogenous nucleic acids encoding β-ketothiolase and acetoacetyl coenzyme A reductase are expressed in a sufficient amount to produce propylene. In a specific embodiment, β-ketothiolase is encoded by phaA or phbA. In another specific embodiment, acetoacetyl coenzyme A reductase is encoded by phaB, hbd, or phbB. Exemplary β-ketothiolase and acetoacetyl coenzyme A reductase amino acid sequences are provided in Tables 2 and 3, respectively.

Table 2: Exemplary β-ketothiolase amino acid sequences

Genbank Gene Name/ Amino Acid Sequence (SEQ ID NO:#)

Accession # Organism

K96243] GPVPASRRCLERAGWSVGDLDLMEINEAFAAQALAVHKQMGWDTSKVNVNGGAIAIG

HPIGASGCRILVTLLHEMLKRDAKRGLASLCIGGGMGVALALERP (SEQ ID NO:77) acetyl-CoA MTTEIVIVSAARTAVGSFNGAFGATPAHELGAVAVKAAIERAGLAPADIDEVILGQVLGA gi 129644557

acetyltransfe AQGQNPARQAAIKAGVPQEKTAFGINQVCGSGLRAVALAAQQIQAGDASVIVAGGQES 5 rase MSLSQHSAHMRAGTKMGDVKFVDTMIVDGLTDAFNNYHMGITAENVAAKWQISRAE

[Methylosinu QDAFAVASQNKAEAAQKAGKFKDEIVPFTVSTRKGDVIVDQDEYIKHGVTLEGVAKLKP s AFTKEGTVTAANASGLNDGAAALVVMSAAEAARRGLTPLARIASWATAGVDPSVMGS trichosporiu GPIPASRKALEKAGWKIGDLDLVEANEAFAAQALAVNKDLGWDPAIVNVNGGAIAIGHP m OB3b] IGASGARVLTTLLYELARRGGKRGLATLCIGGGMGVALTIER (SEQ ID NO:78) acetyl-CoA MSAVDPIVIVGAARTPIGALLGELKNATAPQLGAAAIRAATERAGLAPERVDDVLMGCVL gi 129644496

acetyltransfe SAGLGQAPARQAALGAGLADTTGCVTINKMCGSGMKALMLAHDQLLAGSSRAIVAGG 6 rase MESMSNAPYLLGRARVGYRMGHGRLIDHMFLDGLEDAYDEGKLMGAFAEDCATTHQF

[Methylosinu TREKQDDYATASLRRAQQAAASGAFDWETTPVATHDRKTSATVTRDELPASAKIENIASL s KPAFRDGGTVTAANSSAISDGAAALALMRRSEAERASLAPLAIVRAHATHAGPPHLFPIA trichosporiu PIGAIAKLCERAGWPLTSVDLFEINEAFAVVVLAAMRELYLPHEKVNVHGGACALGHPIG m OB3b] ASGARIVVTLLAALRKYDLRRGVAALCIGGGEATAMAIETIV (SEQ ID NO:79) acetyl-CoA MPEAYIYDAVRTPRGRGKPNGSLHEVSSLGLAVAALSALKQRNRLDGAPVDDVILGCVD gi 129644550

acetyltransfe PVGEAGGDIARAAAIASGFGYEVPGVQINRFCASGLDAVNFAAAQIMSGQHELTIGGGV 4 rase ESMSRVGIGASGGAWPADPAIAIPSYFMPQGVSADLIATKYGFSRNDVDAYAMQSQQR

[Methylosinu AARAWEEQRFARSVTPVKDVNGLTILDHDEHMRPSTDMQSLGALKPAFAFLAEQAGFD s AVAIQAHPDVEKINYVHHAGNSSGIVDGAAAVLLGSKEAGEKAGLTPRARIRAFANIGSE trichosporiu PALMLTGPVDVTKKLLAKAGMTFGDIDLVEVNEAFAAVVLRFLQAFSLDDSKVNVNGGA m OB3b] IALGHPLGATGAMLVGTALDELERSGKGVALVTLCIGAGMGTATIIERV (SEQ ID

NO:80)

gi 1 23502628 phbA-1 gene MSDPKSIVIASAARTAVGAFNGAFANVPAHELGAVAIKAALERAGVDAADVDEVILGQV product LTAGEGQNPARQAAMGAGCPKETTAFAINQLCGSGLRAVALGMQQIVSGDAKIIVAGG [Brucella suis QESMSMAPHCAYLRSGVKMGDFKMIDTMLKDGLTDAFHGYHMGITAENIARQWQLS 1330] RSEQDEFALASQHKAEAAQKAGRFDEEIVPFTVKARKGDVVVSADEYIRPGTTMEVLAKL

KPAFDKEGTVTAGNASGINDGAAAVVLMDAGEAARRGVKPLARIVSWATAGVDPSIM

GTGPIPATRKALEKAGWSVGDLDLVEANEAFAAQSCAVVRDLGLNPEIVNVNGGAIA IG

HPIGASGARVLTTLLYEMERRDAKRGLATLCIGGGMGVALCVERD (SEQ ID NO:81) gi 126068268 acetyl-CoA MGVMNMREVVIASAARTAVGSFGGAFKSVSAVELGVTAAKEAIKRANITPDMIDESLLG 3 acetyltransfe GVLTAGLGQNIARQIALGAGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDADIMLVGG rase AENMSMSPYLVPSARYGARMGDAAFVDSMIKDGLSDIFNNYHMGITAENIAEQWNIT

[Clostridium REEQDELALASQNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDKDEYIKPGTTMEKLAKL difficile RPAFKKDGTVTAGNASGINDGAAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKIMGY

CD196] GPVPATKKALEAANMTIEDIDLVEANEAFAAQSVAVIRDLNIDMNKVNVNGGAIAIGHPI

GCSGARILTTLLYEMKRRDAKTGLATLCIGGGMGTTLIVKR (SEQ ID NO:82) gi 1 14790401 acetyl-CoA MNSGEETVVIISAARTPIGSFNGALSTLPAHTLGSTVIKEVLKRATIKPEEVSEVIFGQV LTA 4 acetyltransfe GAGQNPARQASVAAGVPYSIPAWSCQMICGSGLKAVSLGAQSIKTGEADIVVAGGMEN rase 2 MSQAPHLVHMRAGVKAGDVSLQDSIICDGLNDAFYKYHMGITAENVAKQWQITREEQ [Xenopus DQLAVQSQNRTEAAQKAGYFDKEIVPVSVPSRKGPVEVKVDEFPRHGSNVEAMSKLKPY laevis] FLKDGSGTVTPANASGINDGAAAVVLIKESEARRRGLTPMARIVASAQAGLDPSIMGVG

PIAAIRKAVEKAGWSLDDIDLFEINEAFAAQALAVVKDLGLNPEKVNCQGGAVALGHPLG

MSGCRILVTLLYALERTGGKKGVAALCIGGGMGIAMCVERTS (SEQ ID NO:83) gi 135844773 acetyl-CoA MRDVVIVAARRTAIGTFGGGLSSLSADQLGTAVIKALMEETGVAGDQINEVVLGQVLTA 7 acetyltransfe GVGQN PARQSAI NAG 1 PASVPAMTI N KVCGSG LKAVH M AVQAI RCGDAE M M 1 AGGQE rase SMSQAPHVLPNSRNGQRMGNWSMVDTMIKDGLWDAFNDYHMGITAENIVEKYGISR

[Marinobacte DEQDEFAAASQQKAAAAREAGYFDGQIVPVSIPQRKGDPIVVDRDEGPRDGVTAEGLG r KLRAAFKKDGTVTAGNASSLNDGAAAVMVCSAEKAKELGLTPIATIKAYANAGVDPTIM manganoxyd GTGPIPASQRCLKLAGWSTEDLDLVEANEAFAAQAISVNRDMGWDTGKVNVNGGAIAL ans Mnl7-9] GHPIGASGCRILVSLLHEMVRRDVHKGLATLCIGGGMGVALAVER (SEQ ID NO:84) gi 129401168 acetyl-CoA MPLSDIVITAAKRTAVGGFMGAFGSTPAHELGRTAILAALAQAGVAPEEVDEVILGQVLT 4 C- AGQGQNPARQAAVNAGIPVERTAIGVNQLCGSGLRAVALAAQAIRAGDARIMVAGGQ Genbank Gene Name/ Amino Acid Sequence (SEQ ID NO:#)

Accession # Organism

acetyltransfe ESMSLAPHAQYLRGGAKMGPISLVDTMTHDGLTDAFNNYHMGITAENLAEKYQISREA rase QDVFSVGSQNKAEAARASGRFKDEIAPVTVKGRKGDTIVDTDEYIRAGATLEAMQSLRP

[Sphingobiu AFRKDGTVTAANASGINDGAAALVLMSAEEAAKRDALVLARIASFATCGVDPSIMGIGP m japonicum APASRQALAKAGWSLADLDLIEANEAFAAQALAVGQELGWDAEKVNVNGGAIAIGHPI UT26S] GASGARVLTTLIYEMQKRDAKKGLATLCIGGGMGVAMCIER (SEQ ID NO:85) gi 121269656 hypothetical MTRKVVIASAARTPVGSFGGALKSQSAADLGIVAAKAAIERAGIKPEDIDETVLGCVLQA G 7 protein LGQNVARQISLGAGIPETTPAMTINKVCGSGLRTVSLAAQMILAGDVDVVLAGGAESMS

ANHYDRO_0 NAPFLLNEARWGARMGNKKLVDEMITDGLWDVYNDYHMGVTAENVAEKYGITREMQ 1105 DDLAAVSQQRASKARAEGRFKDEIAPVEIKDRKGNVTVVEDDEYIRDGVTQEGISKLRPA

[Anaerococc FIKDGTVTAANASGINDGAACLVVMSEEKAKELGVKPLATIVSYASAGVDPKVMGTGPIP us SSKKALEKAGWKVEDLDLVESNEAFAAQSYAVRNEMGFDPEKTNVNGGAIAIGHPIGGS hydrogenalis GARILTTLLFEMQKRDSKKGLATLCIGGGMGTALVVER (SEQ ID NO:86) DSM 7454]

gi 135172876 acetyl-CoA MEDIVIVSAARTAVGKFGGALAKTPATELGAIVIREAIARAGLSSDQIGEVIMGQVLAAG V 0 acetyltransfe GQNPARQASMKAGVAKETPALTINAVCGSGLKAVMLAAQAVAWGDSEIVVAGGQES rase MSLAPHVLPGSRDGQRMGDWKLIDTMIVDGLWDVYNQYHMGITAENVAKAHGITRE

[Acidovorax MQDALALGSQQKAAAAQDAGKFVDEIVGVSLAQKKGDPILFNADEYLNRKTNAEALAG radicis N35] LRPAFDKAGSVTAGNASGLNDGAAAVVVMSAKKAAALGLKPLARIAAFGTSGLDPATM

GMGPVPASRKALQRAGWNAADVDLFELNEAFAAQACAVNKELAIDPAKVNVNGGAIA

IGHPIGASGCRILVTLLHEMQRRDAKKGLAALCIGGGMGVSLALER (SEQ ID NO:87) gi 129568783 acetyl-CoA MSDVVIVSAARTPVGSFNGALSSLPASELGRVAIEAAISRAGLQPSDVDEVILGQVLQAG 5 acetyltransfe AGQGPARQASVKAGIPVESPAWSLNQLCGSGLRAVALAAQQIAAGDAAVVVAGGQES rase MSQAPHAQNLRGGQKMGDLSFVDTMIKDGLWDAFHGYHMGQTAENIASRWQITRA

[Caulobacter DQDAFAVASQNKAEAAQKAGKFDAEIAPVTIKGRKGDTVVDKDEYIRHGVTLESISGLKP segnis ATCC AFTKEGSVTAANASGLNDGAAALVLMSAEEAQKRGLKPLARIASWANAGVEPEIMGTG 21756] PIPASKKALEKAGWTVADLDLVESNEAFAAQSLCVVRELGLDPAKVNVNGGAIAIGHPIG

ASGARVLTTLLHEMKRSGAQKGLATLCIGGGMGVAMCVEAV (SEQ ID NO:88) gi 137499637 unnamed MRDVVIVSAVRTPVGSFCGALGQIPAAELGAIAVKEAINRAGITPEQVDEVILGNVLQAG 4 protein LGQN PARQASI KAG 1 PQE VPSWTLN KVCGSGLKTVVCAAQAI MTG DAD 1 VVAGG M EN product MSLAPYVLTKARTGYRMGNDTVIDSMINDGLTDAFNNYHMGITAENIAEQFNISREEQD

[Desulfospor RYSVRSQN RAEAAIIAGKFNEEIVPVSIPQRKGDPVVVSQDEFPRFGATYEAIAKLRPAFKK osinus DGTVTAANASGINDGAAAIVVMAKEKAEELGLTPLATIKSWASAGVDPKIMGTGPIPAS orientis DSM RKALEKAGLSIDDIDVVEANEAFASQTLSVAQGLNLDPEKTNVNGGAIALGHPVGASGTR 765] ILVTLLHEMKRSNAHRGLATLCIGGGQGVALIVER (SEQ ID NO:89) gi 133094156 acetyl-CoA MHDVVIVAATRTAVGSFQGSLASVAAVDLGAAVIRQLLARTGVDGAQVDEVIMGQVLT 4 acetyltransfe AG AGQN PARQAAI KAG LPFSVPAMTLN KVCGSG LKALH LATQAI RCG DAE 111 AGGQE N rase MSLSNYVLPGARTGLRMGHASMVDTMITDGLWDAFNDYHMGITAENLAQQYDISREA

[Pseudomon QDEFAALSQQKALAAIEAGRFVDEITPILIPQRKGDPLSFATDEQPRAGTTAETLAKLKP AF as syringae KKDGTVTAGNASSLNDGAAAVMLMSAARAEQLGLPVLARIAAYANAGVDPAIMGIGPV pv. pisi str. SATRRCLNKAGWSLADLDLIEANEAFAAQSLSVGKELGLDPQKLNVNGGAIALGHPIGAS 1704B] GCRVLVTLLHEMIRRDVKKGLATLCIGGGQGVALALER (SEQ ID NO:90) gi 1 27375337 atoB gene MPMSDDVVIVSAARTPVGSFNGAFATLPAHDLGAVAIKAALERGGIEPGRVSEVIMGQI product LTAAQGQNPARQASIAAGIPVESPAWGVNQLCGSGLRTVALGYQALLNGDSEI VVAGG [Bradyrhizobi QESMSMAPHAQYLRGGVKMGALEFIDTMIKDGLWDAFNGYHMGNTAENVARQWQI urn TRAQQDEFAVASQQKAEAAQKAGKFNDEIVPVTIKTRKGDVVVSADEYPRHGATLDAM japonicum AKLRPAFEKDGTVTAGSASGINDGAAAVVLMTAKQAAKEGKKPLARIVSWAQAGVDPK USDA 110] IMGSGPIPASRAALKKAGWNVGDLDLIEANEAFAAQACAVNKDLGWDTSKVNVNGGAI

AIGHPVGASGARVLVTLLHEMQKRDSKKGLATLCIGGGMGIAMCLARD (SEQ ID NO:91)

gi 1 16373890 Acetyl-CoA MTNVVIASAARTAVGSFGGAFAKTPAHDLGAAVLQAVVERAGIDKSEVSETILGQVLTA 4 C- AQGQN PARQAHINAGLPQESAAWSLNQVCGSGLRAVALAAQHIQLGDAAIVCAGGQE acetyltransfe NMTLSPHAANLRAGHKMGDMSYIDTMIRDGLWDAFNGYHMGQTAENVAEKWQISR rase EMQDEFAVASQNKAEAAQKAGKFADEIAAFTVKTRKGDIIVDQDEYIRHGATIEAMQKL

[Phaeobacter RPAFAKDGSVTAANASGLNDGAAATLLMSADDAEKRGIEPLARIASYATAGLDPSIMGV gallaeciensis GPIYASRKALEKAGWSVDDLDLVEANEAFAAQACAVNKDMGWDPAIVNVNGGAIAIG

BS107] HPIGASGCRVLNTLLFEMKRRDAKKGLATLCIGGGMGVAMCVERP (SEQ ID NO:92) gi 1 7766963 A Chain A, SIVIASAARTAVGSFNGAFANTPAHELGATVISAVLERAGVAAGEVNEVILGQVLPAGEG Genbank Gene Name/ Amino Acid Sequence (SEQ ID NO:#)

Accession # Organism

Unliganded QNPARQAAMKAGVPQEATAWGMNQLCGSGLRAVALGMQQIATGDASIIVAGGMES

Biosynthetic MSMAPHCAHLRGGVKMGDFKMIDTMIKDGLTDAFYGYHMGTTAENVAKQWQLSRD

Thiolase EQDAFAVASQNKAEAAQKDGRFKDEIVPFIVKGRKGDITVDADEYIRHGATLDSMAKLR

From PAFDKEGTVTAGNASGLNDGAAAALLMSEAEASRRGIQPLGRIVSWATVGVDPKVMGT

Zoogloea GPIPASRKALERAGWKIGDLDLVEANEAFAAQACAVNKDLGWDPSIVNVNGGAIAIGHP

Ramigera IGASGARILNTLLFEMKRRGARKGLATLCIGGGMGVAMCIESL (SEQ ID NO:93) gi 121889204 atoB gene MQDVVIVAATRTAVGSFQGSLAGIPAPELGAAVIRRLLEQTGLDAGQVDEVILGQVLTA 1 product GSGQN PARQAAIKAGLPVGVPAMTLNKVCGSGLKALHLGAQAIRCGDAEVIVAGGQEN

[Pseudomon MSLAPYVMPGARTGLRMGHAKLVDSMIEDGLWDAFNDYHMGITAENLAEKYGLSREE as QDAFAAASQQKAIAAIEGGRFRDEITPIQVPQRKGEPLSFDTDEQPRAGTTVEALAKLKP aeruginosa AFRKDGSVTAGNASSLNDGAAAVLLMSAAKAKALGLPVLARIASYASAGVDPAIMGIGP LESB58] VSATRRALDKAGWSLEQLDLIEANEAFAAQSLAVGRELGWDAARVNVNGGAIALGHPI

GASGCRVLVTLLHEMIRRDAKKGLATLCIGGGQGVALTLARD (SEQ ID NO:94) gi 137429277 phbA3 gene MTEVVIAGAARTPIGSFNGALSAVPAHVLGEVAIREALARAKTDAAEVDEVILGQILTAG 7 product QGQNPARQAAVNAGIPVEATAMGINQLCGSGLRAVALGYQAIKNGDADVLVVGGQES

[Azospirillum MSMAPHVMHLRNGTKMGSAELLDTMLRDGLTDAFHGYHMGTTAENVAQKWQLTRE lipoferum EQDAFAAASQQKAEAAQKAG RFKD El VPVTI KG RKG DVVVSDDEYPKH GTTPESLAKLR

4B] PAFSKDGTVTAGNASGINDGAAALVLMTAENAAKRGVTPLARIVSWATAGVDPAIMGT

GPIPASRKALEKAGWTVDDLDLIEANEAFAAQALSVNKDLGWDTSKVNVNGGAIALGH

PVGASGARVLTTLLYEMQKRDAKKGLATLCIGGGMGIALCVQRD (SEQ ID NO:95) gi 1 17546351 phbA gene MTDVVIVSAVRTAVGKFGGSLAKIPAPELGAAVIREALSRAKVAPDQVSEVIMGQVLTAG product SGQNPARQALIKAGLPDMVPGMTINKVCGSGLKAVMLAANAIVAGDADIVVAGGQEN [Ralstonia MSAAPHVLPGSRDGFRMGDTKLIDSMIVDGLWDVYNQYHMGITAENVAKQYGITREA solanacearu QDAFAVASQNKAEAAQKSGRFNDEIVPILIPQRKGDPIAFAQDEFVRHGATLESMTGLKP m G Ml 1000] AFDKAGTVTAANASGLNDGGAAVVVMSAARAKELGLTPLATIRAYANAGVDPKVMGM

GPVPASKRCLSRAGWSVGDLDLMEINEAFAAQALAVHQQMGWDTAKVNVNGGAIAI

GHPIGASGCRILVTLLHEMQKRDAKKGLASLCIGGGMGVALAVERP (SEQ ID NO:96) gi 122119805 Acetyl-CoA MTDVVIVSAARTAVGKFGGSLAKVAAPELGATVIRAVLERAGVKPEQVSEVIMGQVLTA 6 acetyltransfe GSGQN PARQSLIKAGLPSAVPGMTINKVCGSGLKAVMLAANAIVAGDAEIVVAGGQEN rase MSAAPHVLPGSRDGFRMGDAKLVDTMIVDGLWDVYNQYHMGITAENVAKEYGITREE

[Burkholderia QDAFAALSQNKAEAAQKAGRFNDEIVPVSIPQRKGEPLQFATDEFVRHGVTAESLAGLK multivorans PAFAKDGTVTAANASGINDGAAAVLVMSAQKAQALGLTPLARIKAYANAGVDPSVMG

CGD2M] MGPVPASRRCLERAGWTPGDLDLMEINEAFAAQALAVHKQMGWDTSKVNVNGGAIA

IGHPIGASGCRILVTLLHEMVKRDAKRGLASLCIGGGMGVALAVERP (SEQ ID NO:97) gi 1 16385288 acetyl-CoA MAASEDIVIVGAARTPVGSFAGAFGSVPAHELGATAIKAALERAGVSPDDVDEVIFGQVL 2 acetyltransfe TAAAGQNPARQAAIAAGIPEKATAWGLNQVCGSGLRTVAVGMQQIANGDAKVIVAGG rase QESMSLSPHAQYLRGGQKMGDLKLVDTMIKDGLWDAFNGYHMGQTAENVAQAFQL

[Methylobact TREQQDQFAVRSQNKAEAARKEGRFKEEIVPVTVKGRKGDTVVDTDEYIRDGATVEAM erium AKLKPAFAKDGTVTAANASGLNDGAAALVLMSASEAERRGITPLARIVSWATAGVDPKV extorquens MGTGPIPASRKALEKAGWKPADLDLIEANEAFAAQALAVNKDMGWDDEKVNVNGGAI PA1] AIGHPIGASGARVLITLLHELKRRDAKKGLATLCIGGGMGVAMCVERV (SEQ ID NO:98) gi 1 16934417 acetyl-CoA MREVVIASAVRTALGSFGGSLKDVPAVDLGALVIKEALNKAGVKPECVDEVLMGNVIQA 9 acetyltransfe GLGQNPARQAAVKAGLPVEIPSMTINKVCGSGLRCVALAAQMIKAGDADVIVAGGME rase NMSQGPYVLRTARFGQRMGDGKMVDAMVNDALTDAFNGYHMGITAENIAEQWGLT

[Clostridium REMQDEFAANSQIKAEAAIKAGKFKDEIVPVVIPQRKGDPIVFDTDEFPRFGTTAEKLAK L perfringens C RPAFKKDGTVTAGNASGINDGAAALVVMSAEKAKELGVTPICKIVSYGSKGLDPSIMGYG str. JGS1495] PFYATKKALEGTGLKVEDLDLIEANEAFAAQSLAVAKDLEFDMSKVNVNGGAIALGHPVG

ASGARILVTLLHEMMKRDAKRGLATLCIGGGMGTALIVER (SEQ ID NO:99) gi 134586944 acetyl-CoA MSENIVIVDAGRTAIGTFGGSLSSLPATELGTTVLKALLARTGIAPDQIDEVILGQVLTA GV 7 acetyltransfe GQNPARQTTLKAGLPHAVPAMTINKVCGSGLKAVHLAMQAVACGDADIVIAGGQECM rase SQSSHVLPRSRDGQRMGDWKMVDTMIVDGLWDAFNQYHMGVTAENIAKQFGFTRE

[Thiorhodoco AQDTFAAESQQKAEAAIKAGRFKDEIVPVSIPQRKGDPLVVDTDEFPRAGTTAAGLGKLR ecus drewsii PAFDKEGTVTAGNASGINDGAAMVVVMKESKAKELGLKPMARIVAFASAGVDPAIMGT AZ1] GPIPASTKCLEKAGWTPADLDLIEANEAFAAQAMSVNKEMGWDLSKVNVNGGAISLGH

PIGASGARVLVTLLHEMQHRDAKKGLATLCIGGGQGVALAVERL (SEQ ID NQ:100) Table 3 : Exemplary acetoacetyl coenzyme A reductase amino acid sequences

Genbank Gene Name/ Amino Acid Sequence (SEQ ID NO:#)

Accession # Organism

gi 1352104657 acetoacetyl- MTNQAPVAWVTGGTGGIGTAICRSLADAGYLVVAGYHNPDKAKTWLETQRADGYNNI

CoA ELSGVDLSDHNACLEGAREIHDKYGPISVLVNCAGITRDGTMKKMSYEQWYEVLDTNLN reductase SVFNTCRSVIEMMLENGYGRIINISSINGRKGQFGQVNYAAAKAGMHGLTMSLAQETAT [Halomonas KGITVNTVSPGYIATDMIMNIPEKVREAIRETIPVKRYGTPEEIGRLVTFLADKESGFIT GAN sp. HAL1] IDINGGQFMG (SEQ ID NO:110)

gi 1289671313 acetoacetyl- MTSRVALVTGGTGGIGTAICKRLADQGHRVASNFRNEEKARDWQQRMQAQGYAFALF

CoA RGDVASSEHARALVEEVEASLGPIEVLVNNAGITRDTTFHRMTAEQWHEVINTNLNSVF reductase NVTRPVIEGMRKRGWGRVIQISSINGLKGQYGQANYAAAKAGMHGFTISLARENAAFG [Xanthomona VTVNTVSPGYVATDMVMAVPEEVRAKIVADIPTGRLGRPEEIAYAVAFLVAEEAAWITGS s campestris NLDINGGHHMGW (SEQ ID NO:lll)

pv.

musacearum

NCPPB 4381]

gi 1330824321 acetoacetyl- MNTTQRTALVTGGNRGLGAAIARALHDAGHRVIVTHTPGNTTIGQWQQAQATQGYKF

CoA AAYGVDVSNYESTQELARRIHADGHRIDILVNNAGITRDATLRKLDKAGWDAVLRTNLDS reductase MFNVTKPFIDPMVERGWGRIVNISSINGSKGQFGQTNYSAAKAGVHGFTKALAQEVAR [Alicycliphilus KGVTVNTVSPGYLATEMVMAVREDMRQKIIDAIPVGRLGQPDEIAALVAFIASEAAAFM denitrificans TGSNVAMNGGQHMY (SEQ ID NO:112)

K601]

gi 1 146278501 acetoacetyl- MSKVALVTGGSRGIGAAISLALKNAGYTVAANYAGNDEAAQKFTAETGIKTYKWSVADY

CoA DACAEGIARVEAELGPVAVLVNNAGITRDSMFHKMTREQWKEVIDTNLSGLFNMTHPV reductase WSGMRDRKFGRIINISSINGQKGQAGQANYSAAKAGDLGFTKALAQEGARAGITVNAIC [Rhodobacter PGYIGTEMVRAIDEKVLNERIIPQIPVGRLGEPEEIARCVVFLASDDAGFITGSTITANG GQ sphaeroides YFT (SEQ ID NO:113)

ATCC 17025]

gi 1 67458545 phbB gene MSEIAIVTGGTRGIGKATALELKNKGLTVVANFFSNYDAAKEMEEKYGIKTKCWNVADFE product ECRQAVKEIEEEFKKPVSILVNNAGITKDKMLHRMSHQDWNDVINVNLNSCFNMSSSV [Rickettsia MEQMRNQDYGRIVNISSINAQAGQVGQTNYSAAKAGIIGFTKALARETASKNITVNCIAP felis GYIATEMVGAVPEDVLAKIINSIPKKRLGQPEEIARAVAFLVDENAGFITGETISINGGH N

URRWXCal2] Ml (SEQ ID NO:114)

gi 194497737 Acetoacetyl- MSRVAIVTGGTRGIGEAISLALKEMGYAVAANYAGNDEKAKAFTDKTGIAAFKWDVGD

CoA HQACLDGCAQVAEVLGPVDIVVNNAGITRDGVLAKMSFDDWNEVMRINLGGCFNMA reductase KACFGGMRERGWGRIVNIGSINGQAGQYGQVNYAAAKSGIHGFTKALAQEGAKYGVT [Sphingomon VNAIAPGYIDTDMVAAVPAPVLEKIVAKIPVGRLGQAHEIARGVAFFCSEDGGFVTGSTL S as sp. SKA58] INGGQHMY (SEQ ID NO:115)

gi 128901060 acetoacetyl- MKKVALITGSKGGIGSAISSQLVNDGYRVIATYFTGNYECALEWFNSKGFTKDQVRLFEL D

CoA VTNTAECAEKLAQLLEEEGTIDVVVNNAGITRDGVFKKMTAQAWNDVINTNLNSLFNVT reductase QPLFAAMCEKGGGRVINISSVNGLKGQFGQANYSAAKAGMIGFSKALAYEGARSGVTV [Vibrio NVIAPGYTGTPMVEQMKPEVLESITNQIPMKRLATPEEIAASVSFLVSDAGAYITGETLS V parahaemolyt NGGLYMH (SEQ ID NO:116)

icus RIMD

2210633]

gi 1 161522918 acetoacetyl- MSAKRVAFVTGGMGGLGAAISRRLHDVGMTVAVSHTEGNDHVATWLTHEREAGRTF

CoA HAFEVDVADYDSCRQCASRVLAEFGRVDVLVNNAGITHDATFVKMTKSMWDAVLRTN reductase LDGMFNMTKPFVPGMIEGGFGRIVNIGSVNGSRGAYGQTNYAAAKAGIHGFTKALALEL [Burkholderia ARHGVTVNTVAPGYLATAMLETVPKEVLDTKILPQI PVGRLGNPDEIAALVAFLCSDAAAF multivorans ATGAEFDVNGGMHMK (SEQ ID NO:117)

ATCC 17616]

gi 1 161524658 acetyacetyl- MSQRIAYVTGGMGGIGTSICQRLSKDGFKVVAGCGPNSPRRVKWLEEQKALGFDFIASE

CoA GNVGDWDSTKAAFDKVKAEVGEIDVLVNNAGITRDVVFRKMTHEDWTAVIDTNLTSLF reductase NVTKQVIDGMVERGWGRIINISSVNGQKGQFGQTNYSTAKAGIHGFTMALAQEVATKG [Burkholderia VTVNTVSPGYIGTDMVKAIRPDVLEKIVATIPVRRLGTPEEIGSIVAWLASNDSGFATGA D multivorans FSLNGGLHMG (SEQ ID NO:118)

ATCC 17616]

gi 1351728759 3-ketoacyl- MSQKVAYVTGGMGGIGTAICQRLHKEGFKVIAGCGPTRDHAKWLAEQKALGYTFYASV

(acyl-carrier- GNVGDWDSTVEAFGKTKAEHGTIDVLVNNAGITRDRMFLKMSREDWDAVIETNLNSM protein) FNVTKQVVADMVEKGWGRIVNISSVNGEKGQAGQTNYSAAKAGMHGFSMALAQELA Genbank Gene Name/ Amino Acid Sequence (SEQ ID NO:#)

Accession # Organism

reductase TKGVTVNTVSPGYIGTDMVKAIRPDVLEKIVATVPVKRLGEPSEIASIIAWLASEEGGYA TG

[Acidovorax ADFSVNGGLHMG (SEQ ID NO:119)

radicis N35]

gi 1 240140211 phaB gene MAQERVALVTGGTRGIGAAISKRLKDKGYKVAANYGGNDEAANAFKAETGIPVFKFDVG product DLASCEAGIKAIEAELGPIDILVNNAGITRDGAFHKMTFEKWQAVIRTNLDSMFTCTRPL I

[Methylobact EGMRSRNFGRIIIISSINGQKGQAGQTNYSAAKAGVIGFAKALAQESASKGVTVNVVAPG erium YIATEMVMAVPEDIRNKIISTIPTGRLGEADEIAHAVEYLASDEAGFVNGSTLTINGGQH F extorquens V (SEQ ID NO:120)

AMI]

gi 1 148258780 3-oxoacyl- MARVALVTGGTRGIGAAISKALKAAGHKVAANYGGNDAAAEKFKSETEIPVYKWDVSSF

ACP DACAEGIKKVEAELGPVDILVNNAGITRDTAFHKMTLEQWSAVINTNLGSLFNMTRPVIE

reductase GMRARKFGRIINISSINGQKGQFGQVNYSAAKAGDIGFTKALALETAKAGITVNVICPGY I

[Bradyrhizobi NTEMVQAVPKDVLEKAILPLIPVGRLGEPEEIARAVVFLAADEAGAITGSTLSINGGQYM A urn sp. BTAil] (SEQ ID NO:121)

gi 1 126728325 Acetoacetyl- MARVALVTGGSRGIGEAISKALKAEGYTVAATYAGNDEKAAAFTADTGIKTYKWNVADY

CoA ESSKAGIAQVEADLGPIDVVVANAGITRDAPFHKMTPAQWNEVIDTNLTGVFNTVHPV reductase WPGMRERKFGRIIVISSINGQKGQFAQVNYAATKAGDLGIVKSLAQEGARAGITANAICP

[Sagittula GYIATEMVMAVPEKVRESIIGQIPAGRLGEPEEIARCVVFLASDDAGFINGSTISANGAQ F stellata E-37] FV (SEQ ID NO:122)

gi 1 15895965 hbd gene MKKVCVIGAGTMGSGIAQAFAAKGFEVVLRDIKDEFVDRGLDFINKNLSKLVKKGKIEEA product TKVEILTRISGTVDLNMAADCDLVIEAAVERMDIKKQI FADLDNICKPETILASNTSSLSITE

VASATKRPDKVIGMHFFNPAPVMKLVEVIRGIATSQETFDAVKETSIAIGKDPVEVAEAP G FVVNRILIPMINEAVGILAEGIASVEDIDKAMKLGANHPMGPLELGDFIGLDICLAIMDV LY SETGDSKYRPHTLLKKYVRAGWLGRKSGKGFYDYSK (SEQ ID NO:123)

In certain embodiments, a non-naturally occurring CI metabolizing organism according to any of the embodiments disclosed herein is a CI metabolizing bacterium selected from Methylosinus trichosporium strain OB3b, Methylococcus capsulatus Bath strain, Methylomonas methanica 16A strain, Methylosinus

trichosporium (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197),

Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris, Methylacidiphilum infernorum, Methylomicrobium alcaliphilum, or Methylibium petroleiphilum.

In certain embodiments, a non-naturally occurring CI metabolizing organism may be a syngas or CO utilizing bacterium that naturally possesses the ability to utilize syngas or CO, such as Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium

methylotrophicum, Clostridium Woodii, Clostridium neopropanologen Ralstonia eutropha, or Eurobacterium limosum.

In certain embodiments, a non-naturally occurring CI metabolizing organism may be a methylotrophic bacterium, such as Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, or Methylobacterium nodularis.

In another embodiment, the present disclosure provides non-naturally occurring microbial organisms that have been genetically modified with a novel metabolic pathway for producing propylene from a carbon substrate. More specifically, the non-naturally occurring microbial organisms include an exogenous nucleic acid encoding 4-oxalocrotonate decarboxylase and convert a carbon substrate to propylene. As described previously, 4-OD is used in a novel propylene biosynthetic pathway to catalyze decarboxylation of crotonic acid to propylene and C0 ₂ (see Figure 1). Sources of 4-OD encoding nucleic acid molecules may include any species, prokaryotic or eukaryotic, where the encoded gene product is capable of catalyzing decarboxylation of crotonic acid to propylene and C0 ₂ . Exemplary amino acid sequences of 4-OD are shown in Figure 2.

In certain embodiments, the present disclosure provides a non-naturally occurring microbial organism containing an exogenous nucleic acid encoding a 4- oxalocrotonate decarboxylase, wherein the non-naturally occurring microbial organism is capable of converting a carbon substrate into propylene.

In certain embodiments, non-naturally occurring microbial organisms that include an exogenous nucleic acid encoding a 4-oxalocrotonate decarboxylase and convert a carbon substrate into propylene may further include an exogenous nucleic acid encoding crotonase, or further include an exogenous nucleic acid encoding a crotonase and an exogenous nucleic acid encoding a crotonyl thioesterase. Depending on the host microbial organism selected, a microbial organism may or may not have endogenous enzyme(s) that would participate with 4-OD in forming a biosynthetic propylene synthesis pathway. As described in detail previously, crotonase catalyzes the dehydration of 3-hydyroxybutyryl-CoA to crotonyl-CoA. Crotonyl-CoA thioesterase catalyzes the conversion of crotonyl-CoA to crotonic acid. For example, if a host microbial organism selected is deficient in crotonase, then exogenous expression of crotonase can be included in the microbial organism. In another example, if a host microbial organism is deficient in crotonase and a thioesterase capable of converting crotonyl-CoA to crotonic acid, then exogenous expression of crotonase and crotonyl- CoA thioesterase can be included in the microbial organism. However, it is understood that exogenous expression of all of these enzymes of a propylene biosynthetic pathway {i.e., 4-OD, crotonase, and crotonyl-CoA thioesterase) may be included, even if the host microbial organism contains at least one of the propylene pathway enzymes {e.g., crotonase or crotonyl thioesterase). Expression of exogenous nucleic acids encoding enzymes of the propylene biosynthetic pathway disclosed herein is in a sufficient amount to produce propylene. Sources of crotonase and crotonyl-CoA thioesterase encoding nucleic acids may include any species, prokaryotic or eukaryotic, where the encoded gene products are capable of catalyzing dehydration of 3-hydyroxybutyryl- CoA to crotonyl-CoA and conversion of crotonyl-CoA to crotonic acid, respectively. Exemplary amino acid sequences for crotonase and crotonyl-CoA thioesterase are shown in Figure 4 and Figure 3, respectively.

In a further embodiment, non-naturally occurring microbial organisms as described herein do not have a functional PHB synthase or a substantial amount of functional PHB synthase. As described in detail previously, by inhibiting or reducing PHB function in a microbial organism, propylene synthesis yields may be increased by funneling more 3-hydroxybutyryl-CoA into the propylene pathway via conversion to crotonyl-CoA and to crotonic acid and to propylene (see Figure 1).

Additionally, if non-naturally occurring microbial organisms as described herein do not possess an endogenous PHB synthesis pathway, then non- naturally occurring microbial organisms may be genetically modified to include an exogenous nucleic acid encoding β-ketothiolase and an exogenous nucleic acid encoding acetoacetyl-CoA reductase to provide the capability of producing 3- hydroxybutyrl-CoA, substrate for the crotonase enzyme in the propylene synthesis pathway described herein. Non-naturally occurring microbial organisms that do possess an endogenous PHB synthesis pathway may also be genetically modified with an exogenous nucleic acid encoding β-ketothiolase and an exogenous nucleic acid encoding acetoacetyl-CoA reductase to increase expression of these enzymes. In a specific embodiment, β-ketothiolase is encoded by phaA or phbA. In another specific embodiment, acetoacetyl coenzyme A reductase is encoded by phaB or phbB.

Exemplary β-ketothiolase and acetoacetyl coenzyme A reductase amino acid sequences are provided in Tables 2 and 3, respectively.

Nucleic acid sequences encoding for and amino acid sequences for proteins, protein domains and fragments thereof, for proteins described herein, such as 4-OD, crotonase, crotonyl thioesterase, acetoacetyl coenzyme A reductase, or β- ketothiolase, and domains thereof, that are described herein include natural and recombinantly engineered variants. These variants retain the function and biological activity (including enzymatic activities if applicable) associated with the parent (or wildtype) protein. These variants may have improved function and biological activity (e.g., higher enzymatic activity, improved specificity for substrate) than the parent (or wildtype protein). For example, a variant 4-OD enzyme may be engineered with reduced or eliminated decarboxylation activity on 4-oxalocrotonate, but retains or has increased decarboxylation activity on crotonic acid substrate. Conservative

substitutions of amino acids are well known and may occur naturally in the polypeptide (e.g., naturally occurring genetic variants) or may be introduced when the polypeptide is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a polypeptide using well-known and routinely practiced mutagenesis methods (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY 2001). Oligonucleotide-directed site- specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Deletion or truncation variants of proteins may also be constructed by using convenient restriction endonuclease sites adjacent to the desired deletion. Alternatively, random mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide - directed mutagenesis may be used to prepare polypeptide variants (see, e.g., Sambrook et al., supra).

Differences between a wild type (or parent) nucleic acid or polypeptide and the variant thereof, may be determined by methods routinely practiced in the art to determine identity, which are designed to give the greatest match between the sequences tested. Methods to determine sequence identity can be applied from publicly available computer programs. Computer program methods to determine identity between two sequences include, for example, BLASTP, BLASTN (Altschul, S.F. et al, J. Mol. Biol. 215: 403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD.

Assays for determining whether a polypeptide variant folds into a conformation comparable to the non-variant polypeptide or fragment include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of ligand-binding functions, the retention of enzymatic activity (if applicable), and the sensitivity or resistance of the mutant protein to digestion with proteases (see Sambrook et al, supra). Polypeptides, variants and fragments thereof, can be prepared without altering a biological activity of the resulting protein molecule (i.e., without altering one or more functional activities in a statistically significant or biologically significant manner). For example, such substitutions are generally made by interchanging an amino acid with another amino acid that is included within the same group, such as the group of polar residues, charged residues, hydrophobic residues, and/or small residues, and the like. The effect of any amino acid substitution may be determined empirically merely by testing the resulting modified protein for the ability to function in a biological assay, or to bind to a cognate ligand or target molecule.

It is understood that the non-naturally occurring microbial organisms or CI metabolizing organisms that have been genetically modified as described herein may lead to the biosynthetic production of propylene, including other pathway intermediates (e.g., crotonate or crotonyl-CoA) and downstream products. Like other alkenes, propylene undergoes addition reactions relatively easily at room temperature due to the relative weakness of its double bond. Through polymerization, oxidation, halogenations and hydrohalogenation, alkylation, hydration, oligomerization, and hydroformylation reactions, which are well known to a person of skill in the art, propylene may be converted into other downstream products (e.g., polypropylene, propylene oxide). These addition reactions may occur spontaneously in the non- naturally occurring microbial organisms or CI metabolizing organisms, or the organisms may be further genetically modified to add or enhance addition reaction capability (e.g., to increase conversion to polypropylene or propylene oxide). For example, in methanotrophic bacteria that are genetically modified with a biosynthetic propylene pathway, propylene that is produced may spontaneously be oxidized into propylene oxide via a methane-monooxygenase-catalyzed reaction (see, e.g., U.S. Patent Publication 2002/0168733, U.S. Patent Publication 2003/0203456).

Alternatively, the non-naturally occurring microbial organisms or CI metabolizing organisms may comprise further genetic modifications to inhibit or reduce endogenous enzyme activity that catalyze an addition reaction (e.g., to inhibit conversion to propylene oxide). In non-naturally occurring organisms that spontaneously convert or are genetically modified to convert propylene into a downstream product (e.g.

propylene oxide), there may be little propylene product to recover and measure, and the downstream product (e.g., propylene oxide) may be recovered and measured as a surrogate for propylene production.

Methods of Producing Crotonic Acid or Propylene in Non-naturally Occurring C I Metabolizing Organisms

In certain embodiments, the present disclosure provides methods of producing propylene by culturing non-naturally occurring CI metabolizing organisms according to any of the embodiments as described herein (e.g., capable of converting a CI substrate into propylene), under conditions sufficient to produce propylene. In a specific embodiment, the non-naturally occurring CI metabolizing organisms as disclosed herein produce from about 0.1 grams of propylene/L/day to about 50 grams of propylene/L/day. In another embodiment, the non-naturally occurring C I metabolizing organisms as disclosed herein produce about 0.1 g, 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 1 1 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, or 60 g propylene/L/day.

Additionally, the present disclosure provides methods of producing propylene in non-naturally occurring microbial organisms according to any of the embodiments as described herein (e.g., having a partially heterologous propylene biosynthetic pathway), by culturing the non-naturally occurring microbial organisms under conditions sufficient to produce propylene. In a specific embodiment, the non- naturally occurring microbial organisms as disclosed herein produce from about 0.1 grams of propylene/L/day to about 50 grams of propylene/L/day. In another embodiment, the non-naturally occurring microbial organisms as disclosed herein produce about 0.1 g, 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 1 1 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, or 60 g propylene/L/ day.

Also disclosed herein are methods of producing crotonic acid by culturing non-naturally occurring C 1 metabolizing organisms under conditions sufficient to produce crotonic acid, wherein the organisms include an exogenous nucleic acid encoding crotonyl-CoA thioesterase. In a specific embodiment, the non-naturally occurring C I metabolizing organisms as disclosed herein produces from about 0.1 grams of crotonic acid/L/day to about 50 grams of crotonic acid/L/day. In another embodiment, the non-naturally occurring CI metabolizing organism as disclosed herein produces about 0.1 g, 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 1 1 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, or 60 g crotonic acid/L/day.

Codon Optimization

Expression of recombinant proteins is often difficult outside their original host. For example, variation in codon usage bias has been observed across different species of bacteria (Sharp et al, 2005, Nucl. Acids. Res. 33 : 1 141-1 153).

Over-expression of recombinant proteins even within their native host may also be difficult. In certain embodiments of the invention, nucleic acids (e.g. , a nucleic acid encoding 4-OD, crotonyl-CoA thioesterase, or crotonase) that are to be introduced into organisms of the invention may undergo codon optimization to enhance protein expression. Codon optimization refers to alteration of codons in genes or coding regions of nucleic acids for transformation of an organism to reflect the typical codon usage of the host organism without altering the polypeptide for which the DNA encodes. Codon optimization methods for optimum gene expression in heterologous organisms have been previously described (see., e.g., Welch et al, 2009, PLoS One 4:e7002; Gustafsson et al, 2004, Trends Biotechnol. 22:346-353; Wu et al, 2007, Nucl. Acids Res. 35:D76-79; ViUalobos et al, 2006, BMC Bioinformatics 7:285; U.S. Patent Publication 2011/0111413; U.S. Patent Publication 2008/0292918). Transformation Methods

Non-naturally occurring microbial organisms as described herein may be transformed to comprise at least one exogenous nucleic acid to provide the host organism with a new or enhanced activity (e.g., enzymatic activity) or may be genetically modified to remove or substantially reduce an endogenous gene function (e.g., enzymatic activity) using a variety of methods known in the art. Recombinant methods for exogenous expression of nucleic acids in microbial organisms are well known in the art. Such methods can be found described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor

Laboratory, New York (2001); and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

A non-naturally occurring CI metabolizing bacterium; non-naturally occurring obligate CI metabolizing organism; non-naturally occurring CI metabolizing organism, wherein the organism does not include Pichia pastoris; a non-naturally occurring methanotrophic or methylotrophic bacterium; or a non-naturally occurring CO utilizing bacterium as described herein may be transformed to comprise at least one exogenous nucleic acid to provide the host with a new or enhanced activity (e.g., enzymatic activity) or may be genetically modified to remove or substantially reduce an endogenous gene function (e.g., enzymatic activity) using a variety of methods known in the art. While genetic engineering tools of C 1 metabolizing organisms are not as extensive as for other microbial organisms (e.g., E. coli), significant advances have been made allowing genetic manipulation of CI metabolizing organisms, as

summarized below.

Transformation refers to the transfer of a nucleic acid (e.g., exogenous nucleic acid) into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid are referred to as "non-naturally occurring" or "recombinant" or "transformed" or "transgenic" organisms.

Expression systems and expression vectors useful for the expression of heterologous nucleic acids in C 1 metabolizing organisms are known. Vectors or cassettes useful for the transformation of suitable host organisms are available.

Electroporation of CI metabolizing bacteria has been previously described in Toyama et al., 1998, FEMS Microbiol. Lett. 166: 1-7 (Methylobacterium extorquens); Kim and Wood, 1997, Appl. Microbiol. Biotechnol. 48: 105-108

{Methylophilus methylotrophus AS1); Yoshida et al, 2001, Biotechnol. Lett. 23:787- 791 (Methylobacillus sp. strain 12S), and US2008/0026005 (Methylobacterium extorquens).

Bacterial conjugation, which refers to a particular type of transformation involving direct contact of donor and recipient cells, is more frequently used for the transfer of nucleic acids into CI metabolizing bacteria. Bacterial conjugation involves mixing "donor" and "recipient" cells together in close contact with each other.

Conjugation occurs by formation of cytoplasmic connections between donor and recipient bacteria, with unidirectional transfer of newly synthesized donor nucleic acids into the recipient cells. A recipient in a conjugation reaction is any cell that can accept nucleic acids through horizontal transfer from a donor bacterium. A donor in a conjugation reaction is a bacterium that contains a conjugative plasmid, conjugative transposon, or mobilized plasmid. The physical transfer of the donor plasmid can occur through a self-transmissible plasmid or with the assistance of a "helper" plasmid.

Conjugations involving CI metabolizing bacteria have been previously described in Stolyar et al, 1995, Mikrobiologiya 64:686-691; Motoyama et al, 1994, Appl. Micro. Biotech. 42:67-72; Lloyd et al, 1999, Archives of Microbiology 171 :364-370; and Odom et al, PCT Publication WO 02/18617; Ali et al, 2006, Microbiol. 152:2931- 2942.

Expression of heterologous nucleic acid molecules in CI metabolizing bacteria is known in the art (see, e.g., U.S. Patent 6,818,424, U.S. Patent Publication 2003/0003528). Mu transposon based transformation of methylotrophic bacteria has been described (see, e.g., Akhverdyan et al, 2011, Appl. Microbiol. Biotechnol.

91 :857-871). A mini-Tn7 transposon system for single and multicopy expression of heterologous genes without insertional inactivation of host genes in Methylobacterium has been described (see, e.g. U.S. Patent Publication 2008/0026005).

Various methods for inactivating, knocking-out, or deleting endogenous gene function in CI metabolizing organisms may be used. Allelic exchange using suicide vectors to construct deletion/insertional mutants in slow growing CI metabolizing bacteria have also been described in Toyama and Lidstrom, 1998, Microbiol. 144: 183-191; Stolyar et al, 1999, Microbiol. 145: 1235-1244; Ali et al, 2006, Microbiology 152:2931-2942; Van Dien et al, 2003, Microbiol. 149:601-609.

Suitable homologous or heterologous promoters for high expression of exogenous nucleic acids may be utilized. For example, U.S. Patent 7,098,005 describes the use of promoters that are highly expressed in the presence of methane or methanol for heterologous gene expression in CI metabolizing bacteria. Additional promoters that may be used include deoxy-xylulose phosphate synthase methanol dehydrogenase operon promoter (Springer et al, 1998, FEMS Microbiol. Lett. 160: 119-124); the promoter for PHA synthesis (Foellner et al. 1993, Appl. Microbiol. Biotechnol. 40:284- 291); or promoters identified from native plasmid in methylotrophs (EP296484). Non- native promoters include the lac operon Plac promoter (Toyama et al, 1997,

Microbiology 143:595-602) or a hybrid promoter such as Ptrc (Brosius et al, 1984, Gene 27: 161-172). Regulation of expression of an exogenous nucleic acid molecule in the host CI metabolizing organism may also be utilized. For example, an

inducible/regulatable system of recombinant protein expression in methylotrophic and methanotrophic bacteria has been described in US Patent Publication 2010/0221813.

Methods of screening are disclosed in Brock, supra. Selection methods for identifying allelic exchange mutants are known in the art (see, e.g., U.S. Patent

Publication No. 2006/0057726, Stolyar et al, 1999, Microbiol. 145: 1235-1244; and Ali et al, 2006, Microbiology 152:2931-2942.

Culture Methods

A variety of culture methodologies may be used for the C 1 metabolizing organisms described herein. For example, CI metabolizing organisms, particularly methanotrophic or methylotrophic bacteria, may be grown by batch culture and continuous culture methodologies. In certain embodiments, the cultures are grown in a controlled culture unit, such as a fermentor, bioreactor, hollow fiber membrane bioreactor, or the like.

A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to external alterations during the culture process. Thus, at the beginning of the culturing process, the media is inoculated with the desired organism or organism and growth or metabolic activity is permitted to occur without adding anything to the system.

Typically, however, a "batch" culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures, cells moderate through a static lag phase to a high growth logarithmic phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.

The Fed-Batch system is a variation on the standard batch system. Fed- Batch culture processes comprise a typical batch system with the modification that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measureable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases such as C02. Batch and Fed-Batch culturing methods are common and known in the art (see, e.g., Thomas D. Brock, Biotechnology: A Textbook of Industrial Microbiology, 2 ^nd Ed. (1989) Sinauer Associates, Inc.,

Sunderland, MA; Deshpande, 1992, Appl. Biochem. Biotechnol. 36:227, herein incorporated by reference).

Continuous cultures are "open" systems where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in logarithmic phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added and valuable products, byproducts, and waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limited nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to modulate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art, and a variety of methods are detailed by Brock, supra.

Culture media must contain carbon substrates for the C 1 metabolizing organisms. Suitable substrates include, but are not limited to CI substrates such as methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines (methylamine, dimethylamine, trimethylamine, etc.), methylated thiols, or methyl halogens (bromomethane, chloromethane, iodomethane, dichloromethane, etc.). In certain embodiments, a non-naturally occurring CI metabolizing organism of any of the disclosed embodiments is capable of growth on methane, methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, methylated amines, methylated thiols, or methyl halogens as a carbon source.

A culture media may comprise a single C 1 substrate as the sole carbon source for the CI metabolizing organism, or comprise a mixture of two or more CI substrates (mixed CI substrates) as multiple carbon sources for the CI metabolizing organism. Additionally, some CI metabolizing organisms are known to utilize non-Cl substrates, such as glucosamine and a variety of amino acids for metabolic activity. For example, some Candida species can metabolize alanine or oleic acid (Suiter et al., 1990, Arch. Microbiol. 153:485-489). Methylobacterium extorquens AMI is capable of growth on a limited number of C2, C3, and C4 substrates (Van Dien et al, 2003, Microbiol. 149:601-609). Alternatively, a CI metabolizing organism may be engineered with the ability to utilize alternative carbon substrates. Hence, it is contemplated that a culture media may comprise a mixture of carbon substrates, with single and multi-carbon compounds (mixed carbon sources), depending on the C 1 metabolizing organism selected. In certain embodiments, a CI substrate provided in a mixed carbon source may be a primary carbon source for a CI metabolizing organism. A carbon source may be added to culture media initially, provided to culture media intermittently, or supplied continuously. Propylene Separation and Recovery

Propylene or propylene oxide produced by the non-naturally occurring organisms described herein may be dissolved in the liquid phase or present as gas in the headspace of the culture container. Propylene may be mixed with other gases in the headspace, such as 0 ₂ , C0 ₂ , H ₂ 0 vapor, or methane. Methods for recovering propylene from a gas mixture have been previously described, and include for example, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration (see., e.g., Bai et al., 2000, J. Memb. Sci. 174:67-79; Shi et al, 2006, J. Membr. Sci. 282: 115-123; Membranes: Separation of Organic Vapors from Gas Streams, by Ohlrogge and Sturken, Ullmann's Encyclopedia of Industrial Chemistry, 2002, Wiley- VCH Verlag GmbH & Co., KGaA; U.S. Patent 4,348,476; Hou et al, 1984, Applied Microbio. and Biotechnol. 19: 1-4; each of the preceding references are incorporated herein by reference, in their entirety). A person skilled in the art can adapt propylene recovery methods used in fluidic cracking process (see, e.g., U.S. Patent 3,893,905; U.S. Patent 6,308,532; U.S. Patent 6,730,142; U.S. Patent 7,875,758) to recover propylene from a fermentation off-gas mixture.

Measuring Propylene Production

Methods for measuring propylene and propylene oxide production are well known in the art and include HPLC (high performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), GC-FID (gas chromatography-flame ionization detector) and LC-MS (liquid chromatography-mass spectrometry). Methods of measuring propylene and propylene oxide concentration have also been described in, for example, Brown et al., 1963, Anal. Chem. 35:2172-2176; Lin et al., 2000, J. Am. Chem. Soc. 122: 11275-11285; Lee and Hwang, J. Membrane Sci. 73:37-45; U.S. Patent Publication 2010/0197986; U.S. Patent Publication 2003/0203456; U.S. Patent

Publication 2002/0168733; Stanley and Dalton, 1992, Biocatalysis & Biotransformation 6: 163-175; each of which is incorporated herein by reference in its entirety).

Measuring PHB production

In certain embodiments, the non-naturally occurring organisms as described herein do not produce a substantial amount of polyhydroxybutyrate (PHB). As used herein, "not producing a substantial amount of polyhydroxybutyrate" means that an organism produces at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%o, 90%), 95%), 99%) less polyhydroxybutyrate as compared to a wildtype organism that has a polyhydroxybutyrate synthesis pathway. Methods for determining PHB concentration are well known in the art. Braunegg et al., (1978, European J. Appl. Microbiol. Biotechnol. 6:29-37) describe a gas chromatographic method for

determining PHB concentration comprising a mild acid or alkaline methanolysis of PHB directly without previous extraction from the cells, which is followed by gas chromatography of the 3-hydroxybutyric acid methylester. A quantitative staining method for detecting PHB in viable cells has also been described (see., e.g., Tyo et al, 2006, Appl. Environ. Microbiol. 72:3412-3417). Stopped-flow attenuated total reflection FT-IR spectrometry has been used to determine intracellular PHB content in bacteria (see., e.g., Jarute et al, 2004, Anal. Chem. 76:6353-6358). Additional methods for measuring PHB have been described in Huang and Reusch, 1996, J. Biol. Chem. 271 :22196-22202; Henneke et al, 2005, Bioprocess & Biosystems Engineering 27:359- 364; Pieja et al, 2011, Appl. Environ. Microbiol. 77:6012-6019; Taguchi et al, 2001, FEMS Microbiol. Letters 198:65-71.

Additional methods for measuring PHB synthesis may include measuring PHB synthase expression (see., e.g., Langenbach et al, 1997, FEMS

Microbiol. Lett. 150:303-309; Solaiman et al, 2008, J. Ind. Microbiol. & Biotechnol. 35: 111-120) or enzyme activity (see., e.g., Schubert et al, 1988, J. Bacteriol. 170:5837- 5847; Liebergesell et al, 1994, Eur. J. Biochem. 226:71-80; Valentin and Steinbuchel, 1994, Appl. Microbiol. Biotechnol. 40:699-709).

EXAMPLES

EXAMPLE 1

METHYLOSINUS TRICHOSPORIUM METHANOTROPH

Preparation of NMS Media.

MgS0 ₄ . 7H ₂ 0 l .O g

CaCl ₂ . 6H ₂ 0 0.20 g

Chelated Iron Solution (see below) 2.0 ml

KNOs l .O g

Trace Element Solution (see below) 0.5 ml

KH ₂ P0 ₄ 0.272 g

Na ₂ HP0 ₄ . 12H ₂ 0 0.717 g

Purified Agar (e.g., Oxoid L28) 12.5 g

Distilled deionized water 1.0 L

Adjust pH to 6.8. Autoclave at 121°C for 15 minutes. Chelated Iron Solution:

Ferric (III) ammonium citrate* 0.1 g

EDTA, sodium salt 0.2 g HC1 (concentrated) 0.3 ml

Distilled deionized water 100.0 ml

*0.5 g of Ferric (III) chloride may be substituted.

Use 2.0 ml of this chelated iron solution per liter of final medium. Trace Element Solution:

EDTA 500.0 mg

FeS0 ₄ . 7H ₂ 0 200.0 mg

ZnS0 ₄ . 7H ₂ 0 lO.O mg

MnCl ₂ . 4H ₂ 0 3.0 mg

H ₃ BO ₃ 30.0 mg

CoCl ₂ . 6H ₂ 0 20.0 mg

CaCl ₂ . 2H ₂ 0 1.0 mg

NiCl ₂ . 6H ₂ 0 2.0 mg

Na ₂ Mo0 ₄ . 2H ₂ 0 3.0 mg

Distilled water 1.0 L

Autoclave at 121 °C for 15 minutes.

Growth and Conjugations. The procedure for conjugating plasmids from E. coli into methanotrophs was based on the method developed by Martin, H. & Murrell, J. C. (1995), FEMS Microbiol. Lett. 127: 243-248.

Briefly, a mobilizing plasmid to be conjugated was first transformed into

E. coli SI 7-1 using standard electroporation methods. Transformation was confirmed by selection of kanamycin-resistant colonies on LB-agar containing 20 g/mL kanamycin. Transformed colonies were inoculated into LB media containing 20 g/mL kanamycin and shaken overnight at 37°C. A 10 mL aliquot of the overnight culture was then collected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size). The E. coli donor strain was washed on the filter with 50 mL sterile NMS media to remove residual media and antibiotic.

In parallel, a sample of the M. trichosporium OB3b recipient strain was inoculated into lOOmL serum bottles containing 20-50mL NMS media. The headspace of the bottles was then flushed with a 1 : 1 mixture of oxygen and methane, and the bottles were sealed with butyl butyl rubber septa and crimped. The bottles were shaken continuously in a 30°C incubator until reaching an OD600 of approximately 0.3. The cells were then collected on the same filter as the E. coli donor strain. The filter was again washed with 50 mL of sterile NMS media. The filter was placed (cells up) on an NMS agar plate containing 0.02% (w/v) proteose peptone and incubated for 24 h at 30°C in the presence of methane and oxygen. After 24 h, cells were re-suspended in 10 mL sterile (NMS) medium before being concentrated by centrifugation. The pellet was re-suspended in 1 mL sterile NMS media. Aliquots (100 μΐ) were spread onto NMS agar plates containing 10 g/mL kanamycin.

The plates were incubated in sealed chambers containing a 1 : 1 mixture of methane and oxygen maintained at 30°C. The gas mixture was replenished every 2 days until colonies formed, typically after 7-14 days. Colonies were streaked onto NMS plates containing kanamycin to confirm kanamycin resistance as well as to further isolate transformed methanotroph cells from residual E. coli donor cells.

Deletion of phaC. A synthetic cDNA construct of the M. trichosporium

OB3b phaC gene was synthesized, incorporating several stop mutations and frame shifts in the 5' region of the gene. This cDNA construct was cloned into an appropriate vector for conjugation, but lacking an origin of replication that functions in

methanotrophs, and introduced into M. trichosporium OB3b using the methods described above. This technique ensures that any kanamycin resistant M. trichosporium OB3b colonies must have been incorporated into the genome by recombination.

Identification of homologous recombination events is well-established in the art, and typically performed by PCR and sequencing using unique primers in the genome and the vector construct to confirm proper insertion. Homologous

recombinants are then grown in the absence of selective pressure {e.g. , kanamycin) for several generations, and sensitive clones which have lost the resistance marker are identified by replica plating (or equivalent technique). Approximately 50% of sensitive revertants possess the mutated form of the target gene in place of the wild-type version.

Loss of phaC function is confirmed by growing the cells under nitrogen limited conditions and measuring PHB content as described in Pieja A J, Rostkowski KH, Criddle CS, Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria. Microb. Ecol. 2011 Oct; 62(3):564-73. Briefly, the putative knockout clones inoculated into lOOmL serum bottles containing 20-50mL NMS media. The headspace of the bottles was then flushed with a 1 : 1 mixture of oxygen and methane, and the bottles were sealed with butyl rubber septa and crimped. The bottles were shaken continuously in a 30°C incubator until reaching an OD600 of approximately 0.3-0.6 to ensure the cells are in logarithmic phase growth. Cells are collected by centrifugation at 4,816 x g (4,700 rpm) for 8 min, washed once with nitrogen-free NMS media medium, and re-suspended in nitrogen-free NMS medium to induce PHB production. 20-50 mL aliquots of washed cells are then transferred to serum bottles, sealed, and methane and oxygen added as described above. Cultures are then incubated at 30°C on orbital shakers at 150 rpm. Assays for optical density and PHB production are performed every 1 to 2 h for the first 20 h.

PHB concentration determination. PHB concentration is measured directly via gas chromatography. For each sample, 5 to 10 mg of freeze-dried biomass is weighed out on an analytical balance, transferred to a 12-ml glass vial, and sealed with a polytetrafluoroethylene (PTFE)-lined plastic cap. 2 mL of methanol acidified with sulfuric acid (3%, vol/vol) and containing 1.0 g/L benzoic acid and 2 ml of chloroform are added to each vial. The vials are shaken gently and then heated at 100°C for 3.5 h. Once the vials cool to room temperature, 1 ml deionized water is added to each. The vials are subjected to vortex mixing for 30 s and allowed to stand until phase separation is complete. The organic phase is analyzed using an Agilent 6890N gas chromatograph equipped with an HP-5 column [containing (5% phenyl)- methylpolysiloxane; Agilent Technologies] and a flame ionization detector (FID), dl-β- Hydroxybutyric acid sodium salt is used as a standard.

Introduction of Propylene pathway. Selected crotonase (SEQ ID

NO:32), crotonyl-CoA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate

decarboxylase (SEQ ID NO: 10) sequences were codon optimized (see Table 4) and synthesized with appropriate promoters. The genes are then cloned and transformed into the phaC knockout strain as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

Table 4: Codon optimized sequences for M. trichosporium OB3b

Reference Codon optimized sequence (SEQ ID NO:#)

Sequence

TCGTCCACGGCGGTGCTTACTGTGCGTTGGCGGAGATGCTGGCCACGGAAGCCACGGTCG C CGTGGTGCATGAGAAGGGCATGATGGCGGTCGGCCAGTCGAATCACACCAGCTTCTTCCG C CCTGTGAAAGAGGGCCACGTGCGTGCCGAGGCCGTGCGTATTCACGCGGGCTCGACCACG T GGTTCTGGGACGTCAGCCTGCGGGACGACGCGGGTCGCCTCTGCGCCGTGTCGTCGATGT C CATCGCGGTCCGCCCTCGCCGTGAC (SEQ ID NO:125)

4-oxalocrotonate ATGTCCACGACCAGCATCACCCCGGATGAGATCGCCCAGGTGCTGCTGGCTGGCGAGCGC A decarboxylase ACCGCACCGAGGTGGCGCAGTTCTCGGCGAGCCACCCCGACCTCGACGTCCGGACGGCCT A (SEQ ID NO:10) TGCGGCCCAGCGCGCTTTCGTCCAGGCCAAGCTGGATGCGGGCGAGCAGCTCGTCGGCTA T

AAGCTGGGCCTGACCAGCCGCAACAAGCAGCGCGCCATGGGCGTCGACTGCCCGCTGTAT G

GCCGCGTCACGTCCTCGATGCTCGCGACGTATGGCGATCCCATCCCGTTCGACCGCT TCATCC

ATCCGCGCGTCGAATCGGAGATCGCGTTCCTGCTCAAGCAGGATGTGACCGCTCCGG CGACC

GTGTCGTCGGTCCTCGCGGCCACCGACGTCGTGTTCGGAGCGGTCGACGTGCTCGAC TCGC

GCTACGAGGGGTTCAAGTTCACGCTCGAGGATGTCGTGGCCGATAACGCGAGCGCGG GAG

CGTTCTACCTCGGACCGGTCGCCCGTCCGGCCACCGAGCTCCGCCTCGACCTGCTGG GATGC

ATCGTTCGCGTGGACGGCGAGGTCACCATGACCGCCGCTGGTGCGGCCGTCATGGGC CATC

CCGCCGCGGCGGTCGCGTGGCTCGCCAACCAGCTCGCGCTCGAGGGCGAATCGCTGA AGGC

CGGACAGCTGATCTTCTCGGGTGGCGTCACTGCGCCCGTCCCGGTCGTTCCTGGCGG CAGCG

TCACGTTCGAGTTCGATGGCCTGGGCGTCATCGAGGTGGCTGGCGCC (SEQ ID NO:126)

Production of Propylene from methane. /?/zaC-deleted M. trichosporium transformed with a vector containing genes encoding crotonase and 4- oxalocrotonate decarboxylase are inoculated into lOOmL serum bottles containing 20- 50mL NMS media and 10 ug/mL kanamycin. The bottle headspace is flushed with a 1 : 1 mixture of methane and oxygen, and the bottles are sealed with butyl rubber septa and crimped. The bottles are then shaken continuously while being incubated at 30°C. The headspace gas is refreshed every 2 days as above; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 \L Hamilton syringe and injected into a HP5890 GC equipped with a CP- PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector is connected in splitless mode and maintained at 250°C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because methane is the only carbon source provided to the cells, all propylene produced must have been derived from methane. EXAMPLE 2

METHYLOCOCCUS CAPSULATUS BATH STRAIN METHANOTROPH

Growth and Conjugations. The procedure for conjugating plasmids from E. coli into M. capsulatus was based on the method reported in Ali, H. & Murrell, J. C. (2009). Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath.

Microbiology (2009), 155:761-771.

Briefly, a mobilizing plasmid to be conjugated was first transformed into E. coli SI 7-1 using standard electroporation methods. Transformation was confirmed by selection of kanamycin-resistant colonies on LB-agar containing 20 g/mL kanamycin. Transformed colonies were inoculated into LB media containing 20 g/mL kanamycin and shaken overnight at 37°C. A 10 mL aliquot of the overnight culture was then collected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size). The E. coli donor strain was washed on the filter with 50 mL sterile NMS to remove residual media and antibiotic.

In parallel, a sample of the M. capsulatus recipient strain was inoculated into lOOmL serum bottles containing 20-50mL NMS media. The headspace of the bottles was then flushed with a 1 : 1 mixture of oxygen and methane, and the bottles were sealed with butyl rubber septa and crimped. The bottles were shaken continuously in a 45°C incubator until reaching an OD600 of approximately 0.3. The cells were then collected on the same filter as the E. coli donor strain. The filter was again washed with 50 mL of sterile NMS media. The filter was placed (cells up) on an NMS agar plate containing 0.02% (w/v) proteose peptone and incubated for 24 h at 37°C in the presence of methane and oxygen. After 24 h, cells were re-suspended in 10 mL sterile (NMS) medium before being concentrated by centrifugation. The pellet was re-suspended in 1 mL sterile NMS media. Aliquots (100 μΐ) were spread onto NMS agar plates containing 10 g/mL kanamycin.

The plates were incubated in sealed chambers containing a 1 : 1 mixture of methane and oxygen maintained at 45°C. The gas mixture was replenished every 2 days until colonies formed, typically after 7-14 days. Colonies were streaked onto

NMS plates containing kanamycin to confirm kanamycin resistance as well as to further isolate transformed methanotroph cells from residual E. coli donor cells.

Introduction of Propylene synthesis pathway. Note that M.

capsulatus does not have a native PHA pathway, hence no pathway genes {i.e., phaC) need to be deleted. However, phaA and phaB function must be introduced to the cells to provide the substrate for the crotonase enzyme {i.e., 3-hydroxybutryl-CoA). Selected phaA (SEQ ID NO:77), phaB (SEQ ID NO:123), crotonase (SEQ ID NO:32), crotonyl-CoA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO: 10) sequences were codon optimized (see Table 5) and synthesized with appropriate promoters. The genes are then cloned and transformed into M. capsulatus as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

Table 5: Codon Optimized Sequences for M capsulatus

Reference Codon Optimized Sequence (SEQ ID NO:#)

Sequence

crotonase ATGGAACTTAACAATGTGATCCTGGAGAAAGAAGGTAAAGTCGCCGTGGTGACCATTA (SEQ ID NO:32) ATCGCCCCAAGGCCCTGAACGCCCTGAATTCTGACACGCTGAAAGAAATGGACTACGT

GATCGGCGAAATCGAGAACGACTCCGAGGTGCTGGCCGTGATCCTGACCGGCGCAGG

CGAAAAGTCGTTCGTTGCCGGAGCGGATATCTCCGAGATGAAAGAGATGAACACCAT T

GAGGGCAGGAAGTTCGGCATCCTGGGCAATAAAGTCTTTCGCCGGCTCGAGCTCCTG G

AGAAGCCGGTAATTGCCGCCGTTAATGGCTTCGCGCTCGGTGGCGGATGTGAAATCG C

GATGAGCTGCGACATCCGCATAGCGAGTAGTAACGCGCGGTTCGGCCAGCCCGAGGT C

GGCCTGGGCATCACGCCCGGATTCGGTGGCACTCAGCGGCTGTCGCGCCTGGTGGGC A

TGGGGATGGCCAAGCAGCTGATCTTCACCGCGCAGAACATCAAAGCCGACGAAGCCC T

GCGCATAGGGTTGGTGAACAAAGTCGTGGAGCCGAGCGAGTTGATGAACACCGCCAA

AGAGATCGCCAACAAGATCGTCTCGAACGCACCGGTCGCGGTGAAATTGTCGAAGCA G

GCCATCAACCGCGGCATGCAGTGCGATATCGATACCGCCCTCGCCTTCGAGTCGGAA GC

CTTTGGTGAATGCTTCTCCACCGAAGATCAAAAAGACGCCATGACCGCCTTCATAGA GA

AGCGCAAGATCGAGGG 1 1 1 1 AAGAACCGG (SEQ ID NO:129) crotonyl-CoA ATGCATCGGACCAGCAACGGCAGCCACGCCACAGGTGGCAATCTGCCGGACGTCGCTA thioesterase GCCACTATCCGGTCGCCTACGAGCAGACCCTTGATGGGACGGTGGGCTTCGTGATCGA (SEQ ID NO:29) CGAGATGACGCCAGAGCGAGCGACCGCTAGCGTCGAAGTCACCGATACGTTGCGGCA

GCGGTGGGGCCTGGTCCATGGCGGTGCGTATTGCGCGCTTGCCGAAATGCTGGCCACC

GAGGCTACCGTCGCCGTCGTCCACGAAAAGGGGATGATGGCGGTTGGTCAGTCGAAC C

ATACGTCGTTCTTTCGTCCCGTGAAAGAGGGCCACGTGCGGGCAGAAGCCGTCCGTA TT

CACGCCGGCAGCACCACCTGGTTCTGGGATGTTTCGCTGCGCGATGACGCCGGCAGG C

TGTGCGCCGTCAGTTCCATGTCAATCGCCGTCCGTCCACGCCGGGAT (SEQ ID NO:130)

4-oxalocrotonate ATGTCGACGACGTCCATTACCCCGGACGAGATTGCCCAGGTGCTGCTCGCTGGGGAAC decarboxylase GGAACCGCACCGAAGTGGCCCAGTTCTCCGCGTCCCATCCGGACCTGGATGTTCGCACC (SEQ ID NO:10) GCCTATGCCGCCCAGCGTGC 1 1 1 1 GTCCAGGCCAAGCTGGACGCGGGAGAGCAGCTCG

TCGGCTACAAGCTGGGCCTTACGAGTCGGAACAAGCAGCGTGCCATGGGTGTGGACTG

CCCGCTGTACGGGCGAGTGACGAGCTCTATGCTGGCGACCTACGGGGACCCGATCCC G

TTTGACCGCTTCATCCATCCGCGGGTCGAAAGCGAGATTGCGTTCCTGTTGAAACAG GA

CGTGACCGCTCCGGCCACCGTGTCGTCCGTTCTGGCCGCCACGGACGTCGTCTTTGG CG

CGGTCGACGTACTGGACTCCCGGTACGAAGGCTTCAAGTTCACCCTCGAAGATGTGG T

GGCCGACAACGCCAGCGCTGGCGCGTTCTATCTCGGACCCGTGGCACGTCCCGCTAC C

GAGTTGCGCCTGGACTTGTTGGGGTGCATCGTACGTGTGGACGGCGAAGTCACGATG A

CCGCGGCTGGCGCAGCCGTGATGGGCCACCCGGCAGCGGCAGTGGCCTGGCTCGCGA

ACCAGCTGGCGCTGGAAGGGGAATCCCTGAAAGCCGGTCAACTGATCTTCTCGGGTG G

GGTCACGGCACCCGTCCCTGTGGTGCCTGGCGGATCGGTGACCTTCGAGTTCGATGG C

CTTGGCGTGATCGAGGTGGCCGGAGCA (SEQ ID NO:131)

Production of Propylene from methane. M. capsulatus transformed with the vector described above are inoculated into lOOmL serum bottles containing 20- 50mL NMS media and 10 g/mL kanamycin. The bottle headspace is flushed with a 1 : 1 mixture of methane and oxygen, and the bottles are sealed with butyl rubber septa and crimped. The bottles are then shaken continuously while being incubated at 42- 45°C. The headspace gas is refreshed every 2 days as above; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 \L Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector is connected in splitless mode and maintained at 250°C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because methane is the only carbon source provided to the cells, all propylene produced must have been derived from methane.

EXAMPLE 3

METHYLOBACTE IUM EXTORQUENS METHYLOTROPH

Growth and Transformation. The procedure for introducing plasmids into M. extorquens has been demonstrated in Ueda S., Matsumoto S., Shimizu S., and Yamane T., Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation, Appl. Environ. Microbiol, 1991, April; 57(4): 924-926.

Briefly, wild-type (wt) M. extorquens is cultured at 30°C in NMS media supplemented with 1% methanol. Cells of M. extorquens NR-2 grown to the middle logarithmic phase (1.4 x 10 ⁹ /ml) are harvested by centrifugation at 6,000 x g for 10 min and washed with electroporation buffer (10 mM Tris-HCl, 2 mM MgCl ₂ . 6H ₂ 0, 10% [wt/vol] sucrose [pH 7.5]). Cells are re-suspended in the same buffer at a cell concentration of 7.0 x 10 ¹⁰ /ml. The cell suspension and the solution of vector (70 g/mL) are mixed at a ratio of 9: 1 (vol/vol) in an Eppendorf tube. The mixture (10 xL) is then transferred into a space between the electrodes of a chamber, where it is equilibrated for 3 min. After being subjected to 10 pulses of a 10 kV/cm electric field for 300 μβεΰ/ρώβε, a 5 \L aliquot of the mixture is transferred to an Eppendorf tube. 0.2 mL of NMS medium is then added to the tube. The cell suspension is then incubated for 2 h at 30°C to allow expression of the antibiotic resistance genes prior to plating on NMS plates containing 1% methanol and 20 g/mL kanamycin.

The plates were incubated at 30°C until colonies formed. Colonies were streaked onto duplicate plates to confirm kanamycin resistance as well as to further isolate transformed methylotroph cells from residual E. coli donor cells.

Deletion of phaC. The deletion of the phaC gene has been described in Korotkova N., Lidstrom M.E., Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C(l) and C(2) compounds in the methylotroph

Methylobacterium extorquens AMI, J. Bacterid. 2001 Feb; 183(3): 1038-46. Briefly, insertion cassettes containing a kanamycin resistance marker were constructed with flanking sequences homologous to the areas flanking the phaC gene in the M. extorquens genome. A tetracycline resistance gene was incorporated elsewhere in the plasmid. Transformants were initially selected for resistance to kanamycin, and then screened for sensitivity to tetracycline to identify potential double cross-over recombination events. Correct insertion into and deletion of the phaC gene was confirmed by PCR.

Loss of phaC function is confirmed by growing the cells under nitrogen limited conditions and measuring PHA content as described in Pieja AJ, Rostkowski KH, Criddle CS, Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria, Microb. Ecol. 2011 Oct; 62(3):564-73.

Introduction of Propylene synthesis pathway. Selected crotonase (SEQ ID NO:32), crotonyl-coA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO: 10) sequences were codon optimized (see Table 6) and synthesized with appropriate promoters. The genes are then cloned and transformed into the phaC knockout strain as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

Table 6: Codon Optimized Sequences for extorquens

Reference Codon Optimized Sequence (SEQ ID NO:#)

Sequence

CGCATCCACGCGGGCTCCACCACCTGGTTTTGGGATGTGTCGCTGCGCGATGACGCAG GCCGCCTTTGCGCCGTGTCCAGCATGTCGATCGCGGTGCGGCCCCGCCGCGAC (SEQ ID NO:133)

4-oxalocrotonate ATGAGCACCACGTCGATCACCCCGGACGAGATCGCGCAGGTGCTGCTGGCAGGCGAG decarboxylase CGCAACCGGACCGAGGTCGCCCAGTTCAGCGCCTCGCACCCGGACCTCGACGTGCGC (SEQ ID NO:10) ACGGCGTATGCTGCGCAGCGGGCGTTCGTGCAGGCCAAGCTCGATGCCGGCGAGCA

GTTGGTCGGCTACAAGCTCGGCCTGACCTCGCGGAATAAGCAGCGGGCCATGGGCGT

CGACTGCCCGTTGTATGGTCGCGTCACCAGCAGCATGCTGGCGACCTACGGCGACCC C

ATCCCCTTCGACCGCTTCATCCATCCGCGCGTCGAATCGGAAATCGCCTTCCTGCTG AA

GCAGGATGTCACCGCCCCGGCCACCGTCTCGTCGGTCCTCGCCGCGACCGACGTCGT T

TTCGGCGCTGTCGACGTGCTGGATAGCCGCTACGAGGGCTTCAAGTTCACGCTGGAA

GATGTGGTCGCGGACAACGCCAGCGCCGGAGCCTTCTACCTCGGTCCCGTCGCCCGT C

CGGCCACGGAGCTCCGGCTCGACTTGCTCGGCTGCATCGTCCGGGTCGACGGCGAGG

TTACCATGACCGCAGCGGGAGCCGCCGTGATGGGCCACCCCGCAGCCGCGGTGGCCT

GGCTCGCCAACCAGCTCGCCCTCGAGGGCGAGTCGCTGAAAGCCGGCCAGCTGATCT

TCAGCGGCGGTGTGACGGCGCCGGTCCCCGTCGTGCCCGGTGGCTCGGTCACCTTCG

AGTTCGACGGACTGGGCGTCATCGAGGTGGCCGGCGCC

(SEQ ID NO:134)

Production of Propylene from methanol. /?/zaC-deleted M. extorquens transformed with a vector containing genes encoding crotonase and 4-oxalocrotonate decarboxylase are inoculated into lOOmL sealed flasks containing 20-50mL NMS media, 125 mM methanol, 50 g/mL rifamycin, and 10 g/mL kanamycin. The flask headspace is flushed with oxygen and sealed to prevent loss of the propylene product. The flasks are then shaken continuously while being incubated at 30°C. The headspace gas is refreshed every day; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 xL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector is connected in splitless mode and maintained at 250°C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because methanol is the only carbon source provided to the cells, all propylene produced must have been derived from methanol. EXAMPLE 4

CLOSTRIDIUM AUTOETHANOGENUM

Growth and transformation. C. autoethanogenum is cultivated anaerobically in modified PETC medium (ATCC medium 1754) at 37° C in modified PETC media.

The modified PETC medium contains (per L) 1 g NH ₄ C1, 0.4 g KC1, 0.2 g MgS0 ₄ x7 H ₂ 0, 0.8 g NaCl, 0.1 g KH ₂ P0 ₄ , 20 mg CaCl ₂ x2 H ₂ 0, 10 ml trace elements solution (see below), 10 ml Wolfe's vitamin solution (see below), 2 g

NaHC0 ₃ , and 1 mg resazurin. After the pH is adjusted to 5.6, the medium is boiled, dispensed anaerobically, and autoclaved at 121° C for 15 min. Steel mill waste gas

(composition: 44% CO, 32% N ₂ , 22% C0 ₂ , 2% H ₂ ) or equivalent synthetic mixtures are used as carbon source. The media has a final pH of 5.9 and is reduced with Cystein-HCl and Na ₂ S in a concentration of 0.008% (w/v).

The trace elements solution consists of 2 g nitrilotriacetic acid (adjusted to pH 6 with KOH before addition of the remaining ingredients), 1 g MnS0 ₄ , 0.8 g Fe(S0 ₄ ) ₂ (NH ₄ ) ₂ x6 H ₂ 0, 0.2 g CoCl ₂ x6 H ₂ 0, 0.2 mg ZnS0 ₄ x7 H ₂ 0, 20 mg CuCl ₂ x2 H ₂ 0, 20 mg NiCl ₂ x6 H ₂ 0, 20 mg Na ₂ Mo0 ₄ x2 H ₂ 0, 20 mg Na ₂ Se0 ₄ , and 20 mg Na ₂ W0 ₄ per liter.

Wolfe's vitamin solution (Wolin et al, 1963, J. Biol. Chem. 238:2882- 2886) contains (per L) 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine hydrochloride, 5 mg thiamine-HCl, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg calcium D-(+)- pantothenate, 0.1 mg vitamin B12, 5 mg p-aminobenzoic acid, and 5 mg thioctic acid.

Growth experiments are carried out in a volume of 100 ml PETC media in plastic-coated 500-ml-Schott Duran® GL45 bottles with butyl rubber stoppers and 200 kPa steel mill waste gas as sole energy and carbon source. Growth is monitored by measuring the optical density at 600 nm (OD600 nm).

Transformation methods for C. autoethanogenum are performed as described in U.S. Patent Publication 2011/0236941.

Briefly, to make competent cells, a 50 ml culture of C. autoethanogenum is subcultured to fresh media for 3 consecutive days. These cells are used to inoculate 50 ml PETC media containing 40 mM DL-threonine at an OD600nm of 0.05. When the culture reaches an OD600nm of 0.4, the cells are transferred into an anaerobic chamber and harvested at 4,700xg and 4°C. The culture is twice washed with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl ₂ , 7 mM sodium phosphate, pH 7.4) and finally suspended in a volume of 600 μΐ fresh electroporation buffer. This mixture is transferred into a pre-cooled electroporation cuvette with a 0.4 cm electrode gap containing 1 μg of the methylated plasmid mix and immediately pulsed using the Gene pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600 μΐ, and 25 μΡ. Time constants of 3.7-4.0 ms are achieved. The culture is transferred into 5 ml fresh media. Regeneration of the cells is monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon Spectrophotometer (Thermo) equipped with a tube holder. After an initial drop in biomass, the cells start growing again. Once the biomass has doubled from that point, the cells are harvested, suspended in 200 μΐ fresh media and plated on selective PETC plates (containing 1.2% Bacto™ Agar (BD)) with 4 μg/μl Clarithromycin. After 4-5 days of incubation with 30 psi steel mill gas at 37°C, 15-80 colonies per plate are clearly visible.

The colonies are used to inoculate 2 ml PETC media containing 4 μg/μl Clarithromycin. When growth occurs, the culture is upscaled into 5 ml and later 50 ml PETC media containing 4 μg/μl Clarithromycin and 30 psi steel mill gas as sole carbon source.

Confirmation of Successful Transformation:

To verify the DNA transfer, a plasmid mini prep is performed from 10 ml culture volume using the QIAprep Spin Miniprep Kit (Qiagen). The quality of the isolated plasmid DNA is sufficient to run a control PCR. The PCR is performed with Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) using standard conditions (95° C. for 5 min; 32 cycles of 95° C. for 30 s, 50° C. for 30 s, and 72° C. for 1 min; 72° C. for 10 min). As a further control, 1 μΐ of each of the partly degraded isolated plasmids are re-transformed in E. coli XL 1 -Blue MRF' Kan (Stratagene), from where the plasmids can be isolated cleanly and verified by restriction digests.

Introduction of Propylene synthesis pathway. Note that C.

autoethanogenum does not have a native PHA pathway, hence no pathway genes need to be deleted. However, phaA and phaB function must be introduced to the cells to provide the substrate for the crotonase enzyme.

Selected phaA (SEQ ID NO:77), phaB (SEQ ID NO:123), crotonase (SEQ ID NO:32), crotonyl-coA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO: 10) sequences were codon optimized (see Table 7) and synthesized with appropriate promoters. The genes are then cloned and transformed into C. autoethanogenum as described above. Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression). Table 7: Codon Optimized Sequences for C. autoethanogenum

Reference Codon Optimized Sequence (SEQ ID NO:#)

Sequence

ATTTAAGAATAGA (SEQ ID NO:137)

crotonyl-coA ATGCACAGAACATCTAATGGATCACATGCAACAGGTGGCAATCTACCAGATGTTGCAAG thioesterase TCATTATCCGGTAGCTTATGAACAGACATTAGATGGAACCGTTGG 1 1 1 1 GTGATAGATGA (SEQ ID NO:29) AATGACTCCAGAAAGAGCTACAGCTTCCGTCGAGGTAACTGATACATTACGTCAGAGGT

GGGGTTTGGTTCATGGTGGAGCATATTGTGCTCTTGCGGAAATGTTGGCTACTGAAGCA ACAGTTGCAGTTGTACATGAAAAAGGTATGATGGCAGTTGGTCAATCTAATCACACCAG C I 1 1 1 1 CAGGCCAGTTAAAGAAGGTCATGTTAGAGCCGAGGCGGTTAGGATACATGCAG GAAGTACAACCTGG 1 1 1 1 GGGATGTTTCTTTAAGAGATGATGCTGGTAGATTATGTGCTG TTAGCAGTATGTCCATTGCAGTAAGACCAAGAAGAGAT (SEQ ID NO: 138)

4-oxalocrotonate ATGAGCACTACTAGTATAACACCAGATGAAATTGCTCAAGTACTATTAGCTGGAGAAAG decarboxylase AAATAGAACAGAAGTAGCACAG 1 1 1 1 CAGCTTCACACCCGGATTTAGATGTAAGAACGG (SEQ ID NO:10) CTTATGCTGCTCAAAGAGCATTTGTTCAAGCAAAACTTGATGCAGGAGAGCAGTTAGTA

GGCTATAAGCTTGGACTTACATCTAGGAATAAACAAAGAGCTATGGGTGTAGATTGCCC

ACTTTATGGAAGAGTTACGTCCTCTATGTTGGCCACATATGGAGATCCAATACCATT CGA

CAGATTCATACATCCTAGAGTTGAGTCTGAAATTGCATTCTTATTGAAACAAGATGT TACT

GCTCCTGCTACAGTATCATCCGTACTTGCTGCAACTGATGTAG 1 1 1 1 1 GGTGCAGTGGAT

G 1 1 1 1 GGATTCAAGATATGAAGGATTTAAGTTTACTCTAGAAGATGTAGTTGCAGATAAT

GCCAGTGCAGGAGC 1 1 1 1 1 ACCTTGGACCTGTTGCTAGACCTGCTACAGAGTTAAGACTT

GATTTACTAGGATGTATAGTTAGAGTTGACGGAGAAGTTACAATGACAGCGGCTGGT GC

CGCTGTTATGGGACACCCTGCTGCTGCTGTAGCATGGTTAGCTAATCAACTTGCACT TGA

GGGTGAAAGCTTGAAGGCAGGTCAGCTTATCTTTAGCGGTGGGGTCACTGCTCCTGT TC

CAGTAGTTCCTGGTGGAAGCGTGACCTTTGAATTTGATGGCCTAGGTGTAATAGAAG TA

GCAGGAGCC (SEQ ID NO:139)

Production of Propylene from carbon monoxide. C. ethanogenum transformed with the vector described above are used to inoculate 2 ml PETC media containing 4 μg/μl Clarithromycin. When growth occurs, the culture is upscaled into 5 ml and later 50 ml PETC media containing 4 μg/μl Clarithromycin and 30 psi steel mill gas as sole carbon source. The bottles are then shaken continuously while being incubated at 37°C. The headspace gas is refreshed every 2 days; however, immediately prior to refreshing the headspace, samples are drawn from both the liquid phase and headspace using a 10 Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector is connected in splitless mode and maintained at 250°C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Note that because carbon monoxide is the only carbon source provided to the cells, all propylene produced must have been derived from carbon monoxide. EXAMPLE 5

ESCHERICHIA CO / HETE OT OPH

Growth and Transformation. Growth and transformation methods for E. coli are well-known in the art. E. coli strains were transformed by electroporation using the appropriate plasmids. A single colony from a fresh transformation was then used to seed an overnight culture grown in Luria Broth (LB) supplemented with 1.5% (w/v) glucose and appropriate antibiotics at 37°C in a rotary shaker (200 r.p.m.).

Antibiotics were used at a concentration of 50 μg/ml for strains with a single resistance marker. For strains with multiple resistance markers, kanamycin and chloramphenicol were used at 25 μg/ml and carbenicillin was used at 50 μg/ml.

Cloning and expression of 4-OD genes. 4-OD genes were identified from BLAST searching of the NCBI database using the Pseudomonas putida 4-OD sequence as a starting sequence. 24 individual 4-OD proteins were chosen for expression studies. The proteins were reverse-translated and codon-optimized for E. coli using commercial methods (DNA2.0, Inc. Menlo Park, CA). The genes were then cloned under control of a T7 promoter and expressed in E. coli BL21 (DE3). Briefly, single colonies were inoculated into 2mL cultures of LB containing 50 g/mL kanamycin and shaken overnight at 37°C. 1 mL of the saturated overnight culture was then inoculated into 200 mL LB containing 50 g/mL kanamycin in a 2L flask. The flasks were then shaken at 37°C for 3-4 hours until an OD600 of 0.5-1.0 was reached. The cultures were induced by addition of ImM IPTG and shaken for additional 3h at 37°C. Cells were harvested by centrifugation and analyzed by SDS-PAGE to confirm protein expression (see Figure 5). Activity in cell lysates was confirmed by measuring decarboxylation activity on 4-oxalocrotonate (see Figure 6). 4-OD lysate preparation:

Lysates were generated by resuspending induced E. coli cell pellet (equivalent to lmL culture) in 0.5 mL lysis buffer (20 mM KHP0 ₄ pH 8.0; 0.3 M KC1; 10% (w/v) glycerol; 0.1% NP-40; 0.5 mg/ml lysozyme; 1 mM PMSF). Cells were sonicated 5 seconds and then centrifuged for 15 min at 15,000 x g, 4°C. The cleared supernatant was assayed immediately or stored at -80°C for later assay.

4-oxalocrotonate decarboxylation activity assay:

The assay buffer comprised 100 mM Tris-HCl pH 7.4; 3.3 mM MgS0 ₄ ; 1 mM 4-oxalocrotonate (the stock solution of 4-oxalocrotonate was pre-equilibrated with a 1 : 100 dilution of E. coli lysate expressing 4-oxalocrotonate tautomerase from Pseudomonas putida (UniProtKB/Swiss-Prot Accession No. Q01468, geneid 87856) to achieve a distribution of keto and enol forms of 4-oxalocrotonate). Total reaction volume was 200 μΐ in a 96 well UV transparent plate and read on a SpectraMax Plate Reader (Molecular Devices). The reaction was initiated by addition of 4-OD containing lysates at 1 : 10 to 1 : 1000 final dilutions. Reactions were run at 25°C. Consumption of substrate was monitored by measuring drop in absorbance at 240 nm for the keto tautomer and at 300 nm for the enol tautomer (see Figure 6).

Crotonate decarboxylation assay:

The assay buffer comprised 100 mM Tris-HCl pH 7.4; 3.3 mM MgS0 ₄ ; 87.2 mM crotonic acid. Reactions were initiated by adding lysates to a final concentration of 1 : 10 to 1 : 100 in 1 ml volume in a TargetDP vial (2 ml total volume). Reactions were incubated from 12 to 72 hours at room temperature. Generated propylene was detected by injection of 0.5 μΐ aqueous phase plus 2 μΐ headspace gas onto a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector was connected in splitless mode and maintained at 250°C. Samples were run with He Gas at 7.3 ml/min as a carrier gas; the oven program was set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min. Propylene was identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0 (see Figure 7). Introduction of Propylene synthesis pathway.

Introduction of the pathway is performed essentially as described in Bond- Watts et al., Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 2011 7: 222-227.

Selected phaA (SEQ ID NO:77), phaB (SEQ ID NO:123), crotonase (SEQ ID NO:32), crotonyl-coA thioesterase (SEQ ID NO:29), and 4-oxalocrotonate decarboxylase (SEQ ID NO: 10) sequences were codon optimized (see Table 8) and synthesized with appropriate promoters. The genes are then cloned and transformed into E. coli strain BL21 (DE3). Transformation is confirmed by resistance of the cells to antibiotic selection, and gene expression is confirmed by northern blot (to confirm RNA transcription), western blot, or ELISA methods (to confirm protein expression).

Table 8: Codon Optimized Sequences for E. coli Reference Codon Optimized Sequence (SEQ ID NO:#)

Sequence

(SEQ ID NO:77) GCCTGGCGAAGATCGCGGCACCGGAGTTGGGCGCCAGCGTTATTCGTGCCGTCCTGGAA

CGCGCAGGTGTGAAACCGGAGCAGGTGAGCGAAGTGATCCTGGGTCAAGTGCTGACCG

CAGGCAGCGGTCAAAACCCGGCACGTCAAGCCTTGATTGCCGCAGGTCTGCCAAACG CT

GTTCCGGGCATGACCATTAACAAAGTGTGTGGTTCTGGTCTGAAAGCGGTGATGCTG GC

TGCGAACGCGGTTGTCGCCGGTGATGCGGAAATTGTGGTCGCGGGTGGCCAGGAGAA T

ATGTCCGCAGCTCCGCACGTGCTGCCGGGCAGCCGTGACGGTTTCCGTATGGGCGAT GC

TAAATTGGTAGATAGCATGATTGTTGACGGCTTGTGGGACGTGTATAACAAATATCA CAT

GGGTATCACCGCGGAAAACGTTGCGAAAGAGTACGGTATCACCCGTGAGGCGCAGGA C

CAGTTTGCCGCACTGAGCCAGAACAAGGCCGAAGCGGCGCAAAAAGCAGGCCG 1 1 1 1 G

ATGATGAGATCGTTCCGATTGAGATTCCGCAGCGTAAAGGTGAACCGCTGCGCTTCG CT

ACCGACGAGTTTGTCCGTCACGGCGTTACCGCCGAATCCCTGGCCTCTTTGAAACCG GCG

TTTGCTAAAGAGGGTACCGTCACCGCGGCAAACGCAAGCGGTATTAACGATGGCGCA GC

AGCTGTCCTGGTTATGTCCGCGAAGAAGGCAGAAGCGTTGGGCCTGGAGCCGCTGGC TC

GCATTAAAGCATATGCCAATGCCGGCGTTGATCCGAGCGTTATGGGCATGGGTCCGG TC

CCGGCAAGCCGTCGTTGCCTGGAGCGTGCAGGCTGGTCCGTTGGCGACCTGGATCTG AT

GGAGATCAATGAAGCCTTCGCAGCGCAGGCGCTGGCAGTGCACAAGCAGATGGGTTG G

GACACCAGCAAGGTTAATGTCAATGGTGGCGCAATCGCCATTGGCCATCCTATCGGT GC

GAGCGGTTGTCGTA 1 1 1 1 GGTTACCCTGCTGCATGAAATGCTGAAACGCGACGCCAAGC

GTGGCCTGGCTAGCCTGTGCATCGGTGGTGGTATGGGTGTGGCGCTGGCGCTGGAAC G

TCCA (SEQ ID NO:140)

phaB ATGAAGAAAGTATGCGTCATCGGTGCGGGCACCATGGGCAGCGGTATTGCGCAGGCGT

(SEQ ID NO:123) TTGCAGCCAAGGGCTTCGAGGTGGTCCTGCGCGATATCAAAGATGAGTTCGTTGATCGC

GGTTTGGACTTCATCAACAAAAACCTGAGCAAGCTGGTTAAGAAGGGTAAGATCGAAGA

GGCGACGAAGGTTGAAATTCTGACCCGCATCAGCGGTACTGTTGACCTGAATATGGC GG

CAGACTGCGATTTGGTTATTGAAGCTGCGGTCGAGCGTATGGACATTAAGAAGCAGA TT

TTCGCCGATCTGGACAACATTTGTAAGCCGGAGACGATTCTGGCGAGCAACACCAGC AG

CTTGAGCATTACCGAGGTGGCCTCTGCCACGAAGCGTCCGGATAAGGTCATCGGTAT GC

ACTTCTTTAACCCGGCTCCGGTGATGAAACTGGTCGAGGTGATCCGCGGTATTGCTA CCA

GCCAAGAAACGTTTGACGCTGTGAAAGAGACGTCGATCGCTATCGGCAAGGATCCGG TT

GAGGTGGCAGAAGCTCCGGG 1 1 1 1 GTGGTGAATCGCATCCTGATCCCGATGATCAACGA

GGCCGTAGGTATCCTGGCCGAGGGTATTGCCTCTGTGGAAGATATCGACAAGGCGAT GA

AACTGGGTGCTAATCACCCGATGGGTCCGTTGGAGCTGGGTGACTTCATCGGTCTGG AC

ATTTGTCTGGCGATCATGGACGTTCTGTACTCTGAGACGGGCGACAGCAAATATCGC CCG

CACACCCTGCTGAAAAAGTACGTTCGTGCTGGTTGGCTGGGTCGTAAGTCTGGCAAA GG

CTTCTACGATTACAGCAAG (SEQ ID NO: 141)

crotonase ATGGAGCTGAATAATGTGATTCTGGAGAAAGAGGGCAAAGTCGCTGTTGTTACGATTAA (SEQ ID NO:32) CCGCCCGAAGGCATTGAACGCCCTGAACAGCGATACCCTGAAAGAGATGGATTACGTGA

TTGGCGAGATCGAAAACGACAGCGAAGTTCTGGCCGTCATTCTGACTGGTGCCGGTGAA

AAGAGCTTTGTCGCGGGTGCAGATATTAGCGAGATGAAAGAGATGAATACGATCGAA G

GTCGTAAATTCGGTATCCTGGGCAATAAAGTCTTTCGTCGTTTGGAACTGCTGGAGA AAC

CTGTCATCGCTGCCGTGAATGGCTTCGCGCTGGGCGGTGGCTGCGAGATTGCAATGA GC

TGCGATATCCGTATCGCGAGCAGCAATGCGCGTTTCGGTCAACCGGAAGTGGGTCTG GG

TATCACGCCGGG 1 1 1 1 GGTGGCACCCAACGCCTGAGCCGTTTGGTTGGCATGGGTATGG

CAAAACAACTGATCTTTACCGCGCAGAACATCAAAGCAGATGAAGCTCTGCGCATTG GCT

TGGTCAATAAGGTGGTTGAGCCGAGCGAACTGATGAACACGGCGAAAGAGATCGCGA A

CAAGATCGTGAGCAATGCACCGGTGGCCGTCAAACTGAGCAAACAGGCCATCAATCG TG

GTATGCAATGTGATATCGACACCGCGCTGGCATTCGAAAGCGAGGCATTTGGTGAGT GC

TTCAGCACGGAAGATCAAAAGGATGCAATGACGGCGTTCATTGAAAAACGTAAGATT GA

AGGCTTCAAGAACCGC (SEQ ID NO:142)

crotonyl-coA ATGCATCGTACGAGCAACGGCAGCCACGCCACCGGTGGCAATCTGCCTGATGTCGCGAG thioesterase CCATTATCCGGTTGCATACGAACAGACCCTGGACGGCACCGTCGGTTTCGTGATTGACGA Reference Codon Optimized Sequence (SEQ ID NO:#)

Sequence

(SEQ ID NO:29) AATGACTCCGGAGCGTGCCACCGCGAGCGTTGAGGTCACCGATACCCTGCGCCAGCGTT

GGGGTCTGGTTCATGGTGGTGCATATTGTGCGTTGGCAGAGATGTTGGCGACTGAAGCG ACCGTCGCAGTAGTCCATGAAAAGGGCATGATGGCGGTGGGCCAAAGCAATCACACCA GC I 1 1 1 1 CCGTCCGGTTAAAGAGGGCCATGTTCGCGCAGAGGCGGTGCGTATTCACGCG GGTAGCACGACCTGGTTCTGGGACGTTAGCCTGCGCGATGACGCAGGTCGTCTGTGTGC AGTTAGCAGCATGTCTATTGCTGTCCGTCCGCGTCGCGAC (SEQ ID NO:143)

4-oxalocrotonate ATGAGCACCACGAGCATTACCCCGGACGAAATCGCGCAGGTTCTGCTGGCAGGCGAGC decarboxylase GTAATCGCACCGAGGTGGCGCAA 1 1 1 1 1 GCGTCGCACCCGGATTTGGATGTCCGTACCG (SEQ ID NO:10) CGTACGCGGCACAACGTGCGTTCGTTCAGGCTAAACTGGACGCAGGTGAACAACTGGTC

GGTTACAAATTGGGTCTGACGTCTCGTAATAAGCAGCGCGCAATGGGTGTCGACTGCCC

GCTGTACGGCCGTGTTACCAGCAGCATGCTGGCCACCTATGGTGATCCGATTCCGTT TGA

CCGCTTTATTCACCCACGTGTGGAGTCCGAAATTGCGTTCCTGCTGAAGCAAGATGT GAC

CGCACCGGCGACGGTCAGCAGCGTTCTGGCAGCGACTGACGTCGTGTTTGGCGCGGT TG

ACGTCCTGGATAGCCGTTACGAGGGCTTCAAGTTCACCCTGGAAGATGTTGTTGCAG AC

AATGCATCTGCGGGTGCTTTCTATCTGGGTCCAGTTGCACGTCCAGCGACGGAGCTG CGT

CTGGATCTGCTGGGTTGCATTGTGCGCGTCGACGGCGAAGTGACCATGACCGCAGCG GG

TGCCGCGGTTATGGGCCACCCGGCAGCGGCCGTTGCGTGGCTGGCCAATCAGCTGGC CC

TGGAAGGTGAAAGCCTGAAGGCGGGTCAGCTGATCTTCAGCGGTGGTGTCACTGCGC C

GGTCCCGGTTGTGCCGGGTGGCAGCGTGACCTTCGAGTTTGACGGCCTGGGTGTCAT CG

AAGTGGCAGGTGCA (SEQ ID NO:144)

Production of Propylene from glycerol. Overnight cultures of freshly transformed E. coli strains are grown for 12-16 h in Terrific Broth (TB) at 37°C and used to inoculate TB (50 ml) with 1.5% (w/v) glycerol and appropriate antibiotics to an optical density at 600 nm (OD600) of 0.05 in a 250 mi-baffled flask. The cultures are grown at 37°C in a rotary shaker (200 r.p.m.) and induced with IPTG (1.0 mM) and L- arabinose (0.2%> (w/v) (these inducers are dependent on the promoters used for plasmid construction; choice of inducer are apparent to those of skill in the art) at OD600 = 0.35-0.45. At this time, the growth temperature is reduced to 30°C, and the culture flasks are sealed with butyl rubber stoppers to prevent propylene evaporation.

Additional glucose (1% (w/v)) is added concurrent with culture sampling after 1 d. Flasks are unsealed for 10 to 30 min every 24 h then resealed after sampling. Samples are drawn from both the liquid phase and headspace using a 10 xL Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector is connected in splitless mode and maintained at 250°C. Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min. Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell. Production of Propylene from glucose. Overnight cultures of freshly transformed E. coli strains are grown for 12-16 h in Terrific Broth (TB) at 37°C and used to inoculate TB (50 ml) with 1.5% (w/v) glucose replacing the standard glycerol supplement and appropriate antibiotics to an optical density at 600 nm (OD600) of 0.05 in a 250 mi-baffled flask. The cultures are grown at 37°C in a rotary shaker (200 r.p.m.) and induced with IPTG (1.0 mM) and L-arabinose (0.2% (w/v)) at OD600 = 0.35-0.45. At this time, the growth temperature is reduced to 30°C, and the culture flasks are sealed with butyl rubber stoppers to prevent propylene evaporation. Additional glucose (1%) (w/v)) is added concurrent with culture sampling after 1 d. Flasks are unsealed for 10 to 30 min every 24 h then resealed after sampling. Samples are drawn from both the liquid phase and headspace using a 10 \L Hamilton syringe and injected into a HP5890 GC equipped with a CP-PoraBOND U 25 m x 0.32 mm column and an FID maintained at 200°C. The injector is connected in splitless mode and maintained at 250°C.

Samples are run with He Gas at 7.3 ml/min as a carrier gas; the oven program is set as follows: hold at 50°C 1.5 min; ramp to 300°C at 10°C/min; hold at 300°C 10 min.

Propylene is identified by retention time compared to pure propylene diluted in air or dissolved in pure H ₂ 0. Samples are also taken to measure optical density of the culture, allowing quantitation of specific propylene production rates per cell.

The disclosure of U.S. Provisional Application No. 61/702,534, filed on September 18, 2012, is incorporated by reference herein in its entirety.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. Provisional Application No. 61/702,534 filed on September 18, 2012, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible

embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.