FIELD OF INVENTION

The present invention relates to the field of genetic engineering oriented to obtain improved microbial enzymes with transfructosylation activity for efficient and cost-effective production of fructo-oligosaccharides. More specifically, the invention is directed towards obtaining mutant FTase family of genes from genus Aspergillus.

BACKGROUND

Fructose oligomers, also known as fructooligosaccharides (FOS) constitute a series of homologous oligosaccharides. Fructooligosaccharides are usually represented by the formula GFn and are mainly composed of 1-kestose (GF2), nystose (GF3) and P-fructofuranosylnystose (GF4), in which two, three, and four fructosyl units are bound at the P-2,1 position of glucose.

Fructooligosaccharides (FOS) are characterized by many beneficial properties such as low sweetness intensity and usefulness as a prebiotic. Due to the low sweetness intensity (about one-third to two-third as compared to sucrose) and low calorific values (approximately 0-3 kcal/g), fructooligosaccharides can be used in various kinds of food as a sugar substitute. Further, as a prebiotic, fructooligosaccharides have been reported for being used as protective agents against colon cancer, enhancing various parameters of the immune system, improving mineral adsorption, beneficial effects on serum lipid and cholesterol concentrations and exerting glycemic control for controlling obesity and diabetes (Dominguez, Ana Luisa, et al. "An overview of the recent developments on fructooligosaccharide production and applications." Food and bioprocess technology 7.2 (2014): 324-337.)

However, fructooligosaccharides are found only in trace amounts as natural components in fruits, vegetables, and honey. Due to such low concentration, it is practically impossible to extract fructooligosaccharides from food.

Attempts have been made to produce fructooligosaccharides through enzymatic synthesis from sucrose by microbial enzymes with transfructosylation activity. However, the major constraints in the previous attempts have been the lower catalytic efficiency, feedback inhibition of the enzyme by glucose leading lower FOS yields and the requirement of longer time periods for conversion of sucrose by the enzymes expressed in the recombinant host system. Further, industrial production of microbial enzymes exhibiting transfructosylation activity is challenging due to additional limitations associated with large scale expression of enzyme, enzyme stability, fermentation and purification processes.

Commercial-scale production of fructooligosaccharides requires identification and mass production of efficient enzymes. Due to the aforesaid limitations, the production of microbial enzymes with efficient transfructosylation activity is a costly affair which in-tum increases the production cost of fructooligosaccharides.

Thus, there is a long -felt and continuous need for identifying and providing efficient, cheap and industrially useful enzymes with superior transfructosylation activity, which in turn lowers the cost of production of fructooligosaccharides.

FTase family of genes/proteins includes several enzymes, such as, but not limited to P- fructofuranosidases from Aspergillus niger, fructosyltransferases from Aspergillus japonicus, Arabinanase/levansucrase/invertase from Aspergillus fijiensis and fructosyltransferase from Aspergillus aculeatus, etc. The amino acid sequences of these proteins are represented by Sequence IDs 1, 2, 3 and 4). These enzymes exhibit transfructosylation activity and are thus important in production of fructooligosaccharides.

P-fructofuranosidases and fructosyltransferases are being produced from Aspergillus genus, but there is still requirement of efficient and cost-effective enzymes. The present invention attempts to address the aforesaid problems in prior art by generating various mutant and variant strains so that better enzymes can be obtained in good quantities.

SUMMARY OF THE INVENTION

Technical Problem

The technical problem to be solved in this invention is to provide novel enzymes with superior transfructosylation activity.

The solution to the problem

The problem has been solved by employing unique enzyme engineering as well as bioinformatics based approaches for developing mutants of FTases from genus Aspergillus. Overview of the invention

The present invention relates to nucleic acids, peptide sequences, mutant proteins, vectors and host cells for recombinant expression of a novel FTases, such as, but not limited to P-fructofuranosidase or fructosyltransferase enzymes. Various mutants, such as but not limited to point mutants and deletion mutants as well as combinations thereof are presented herein. The invention also relates to a process for the expression of a novel recombinant FTase mutants as a secreted protein. The enzymes exhibits high purity after filtration, which eliminates the need for costly chromatographic procedures.

Finally, the enzymes can be used for obtaining a high yield of fructooligosaccharides.

BRIEF DESCRIPTION OF DRAWINGS

The features of the present disclosure will become fully apparent from the following description taken in conjunction with the accompanying figures. With the understanding that the figures depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope, the disclosure will be described further through the use of the accompanying figures.

Figure 1 depicts the multiple sequence alignment of the native FTases from some representative Aspergillus genera.

Figure 2a depicts the multiple sequence alignment of the native fop A gene with modified fop A gene.

Figure 2b depicts the multiple sequence alignment of the native// gene with modified// gene. Figure 3 represents the construction scheme of pPICZaA vector.

Figure 4 depicts the results of the restriction digestion analysis performed on the recombinant plasmid pPICZaA containing (a): V44L; (b): T155R; (c): T293W and R286V; (d): T459R; (e): R199K; (f): F182P; (g): 32-654 aa, 32-194 aa and 1-194 aa mutants.

Figure 5 depicts the SDS-PAGE analysis for screening protein expression in Pichia pastoris recombinant strains having integration (a): pPICZaA-V44L; (b): pPICZaA-T155R; (c): pPICZaA-T293W; (d): pPICZaA-R286V; (e): pPICZaA-R199K; (f): pPICZaA-T459R; (g): pPICZaA-F182P.

Figure 6 depicts the SDS-PAGE analysis of samples collected at different time intervals during fermentation trail of various mutant FTase proteins, (a): pPICZaA-V44L; (b): pPICZaA- T155R; (c): pPICZaA-T293W; (d): pPICZaA-R286V; (e): pPICZaA-R199K; (f): pPICZaA- T459R; (g): pPICZaA-F182P

Figure 7 depicts TLC showing formation of FOS from sucrose by the mutant enzymes, (a): V44L; (b): T155R; (c): T293W; (d): R286V; (e): R199K; (f): T459R; (g): F182P

Figure 8 depicts HPLC analysis chromatographs of FOS samples formed by mutant enzymes, (a): V44L; (b): T155R; (c):T293W; (d): R286V; (e): R199K; (f): T459R; (g): F182P Figure 9 depicts the results of the restriction digestion analysis performed on the recombinant plasmid pPICZaA containing (a): N32R, H43Y, H43V, H43L, V125F, V127A, P131Y; (b): F182R, F182A, F182D, F182V, F182T, T293F, T293H; (c): T293R, T293L, T293S, Q327L; (d): Q327N, V343F

Figure 10 depicts the SDS-PAGE analysis for screening protein expression in Pichia pastoris recombinant strains having integration (a): pPICZaA-H43Y; (b): pPICZaA-H43L; (c): pPICZaA-Q327N; (d): pPICZaA- V343F

Figure 11 depicts workflow to identify stabilizing point mutations in FTase

Figure 12 depicts comparative sequences of ft gene and fop A gene

BRIEF DESCRIPTION OF SEQUENCES AND SEQUENCE EISTING

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any vectors, host cells, methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the vectors, host cells, methods and compositions, representative illustrations are now described.

Where a range of values are provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. The term “host cell(s)” includes an individual cell or cell culture which can be, or has been, a recipient for the subject of expression constructs. Host cells include progeny of a single host cell. Host cells for the purposes of this invention refers to any strain of Pichia pastoris which can be suitably used for the purposes of the invention. Examples of strains that can be used for the purposes of this invention include wild type, mut+, mut S, mut- strains of Pichia such as KM71H, KM71, SMD1168H, SMD1168, GS115, X33.

The term “recombinant strain” or “recombinant host cell(s)” refers to a host cell(s) which has been transfected or transformed with the expression constructs or vectors of this invention.

The term “expression vector” refers to any vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host.

The term “promoter” refers to DNA sequences that define where transcription of a gene begins. Promoter sequences are typically located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription. Promoters can either be constitutive or inducible promoters. Constitutive promoters are the promoter which allows continual transcription of its associated genes as their expression is normally not conditioned by environmental and developmental factors. Constitutive promoters are very useful tools in genetic engineering because constitutive promoters drive gene expression under inducer-free conditions and often show better characteristics than commonly used inducible promoters. Inducible promoters are the promoters that are induced by the presence or absence of biotic or abiotic and chemical or physical factors. Inducible promoters are a very powerful tool in genetic engineering because the expression of genes operably linked to them can be turned on or off at certain stages of development or growth of an organism or in a particular tissue or cell type.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).

The term “transcription” refers to the process of making an RNA copy of a gene sequence. This copy, called a messenger RNA (mRNA) molecule, leaves the cell nucleus and enters the cytoplasm, where it directs the synthesis of the protein, which it encodes. The term “translation” refers to the process of translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that it encodes. In the cell cytoplasm, the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein.

The term “expression” refers to the biological production of a product encoded by a coding sequence. In most cases, a DNA sequence, including the coding sequence, is transcribed to form a messenger-RNA (mRNA). The messenger-RNA is then translated to form a polypeptide product that has a relevant biological activity. Also, the process of expression may involve further processing steps to the RNA product of transcription, such as splicing to remove introns, and/or post-translational processing of a polypeptide product.

The term “modified polypeptide/ polynucleotide” as used herein is used to refer to a polypeptide or polynucleotide encoding FTase selected from but not limited to P- fructofuranosidase and/or fructosyltransferase mutants fused to a signal peptide. The functional variant includes any nucleic acid having substantial or significant sequence identity or similarity to the P-fructofuranosidase and/or fructosyltransferase mutants, as described herein which retains the biological activities of the same.

The term “variant” as used herein in reference to pre-cursor peptides/proteins refers to peptides with amino acid substitutions, additions, deletions or alterations that do not substantially decrease the activity of the signal peptide or the enzyme. Variants include a structural as well as functional variants. The term variant also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

Amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups (Table 1) are examples of amino acids that are considered to be variants for one another:

Table 1: Amino add substitution table

The term “point mutation” refer to a large category of mutations that describe a change in single nucleotide of DNA, such that the nucleotide is switched for another nucleotide, or that nucleotide is deleted, or a single nucleotide is inserted into the DNA that causes that DNA to be different from the normal or wild type gene sequence.

The term “deletion mutation” refers to a type of mutation involving the loss of genetic material. It can be small, involving a single missing DNA base pair, or large, involving a piece of a chromosome.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues joined to each other by peptide bonds or modified peptide bonds. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Likewise, "protein" refers to at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides, and peptides. A protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. "Amino acid" includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration.

The term “signal peptide” or “signal peptide sequence” is defined herein as a peptide sequence usually present at the N-terminal end of newly synthesized secretory or membrane polypeptides which directs the polypeptide across or into a cell membrane of the cell (the plasma membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes). It is usually subsequently removed. In particular said signal peptide may be capable of directing the polypeptide into a cell's secretory pathway.

The term “precursor peptide” as used herein refers to a peptide comprising a signal peptide (also known as leader sequences) operably linked to the respective FTases from genus Aspergillus. The signal peptides are cleaved off during post-translational modifications inside the Pichia host cells and the mature FTase mutants are released into the medium. DETAILED DESCRIPTION OF THE INVENTION

The present invention is described by the following specific embodiments. Those with ordinary skill in the art can readily understand the other advantages and functions of the present invention after reading the disclosure of this specification. Various details described in this specification can be modified based on different requirements and applications without departing from the scope of the currently disclosed invention.

Unless contraindicated or noted otherwise, throughout this specification, the terms “a” and “an” mean one or more, and the term “or” means and/or.

The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

As used herein, biotechnological terms have their conventional meaning as illustrated by the following illustrative definitions.

Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

The foregoing broadly outlines the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying the disclosed methods or for carrying out the same purposes of the present disclosure.

Fructosyl transferase (FT) enzyme is a member of glucose hydrolase 32 family (GH32) which catalyses the production of fructans which are fructose oligosaccharides through a retaining mechanism. Three-dimensional structure of the enzyme FT from Aspergillus japonicus (AjFT) has been solved that provided a structural basis of the substrate binding and catalysis carried out by this enzyme. Among the structures that were solved by Chuankhayan et. al.[l], the apo structure (PDB ID: 3LF7) and the kestose bound (PDB ID: 3LDR) structures of AjFT are relevant for the present study. The present invention discloses nucleic acids, vectors and recombinant host cells for efficient production of biologically active and soluble recombinant FTases, including, but not limited to P-fructofuranosidases from Aspergillus niger, fructosyltransferases from Aspergillus japonicus, Arabinanase/levansucrase/invertase from Aspergillus fijiensis, fructosyltransferase from Aspergillus aculeatus, mutants thereof obtained from genus Aspergillus as a secreted protein. Further, the invention provides a process for commercial- sc ale production of recombinant P-fructofuranosidase and/or fructosyltransferase mutants. As representative, but not restrictive examples, we mention fructosyltransferase, encoded by ft gene of Aspergillus japonicus and P-fructofuranosidase, encoded by fopA gene of Aspergillus niger.

The invention contemplates a multidimensional approach for achieving a high yield of novel recombinant FTases mutants in a heterologous host. The codon optimized gene for FTase selected from but not limited to fructosyltransferase or P-fructofuranosidase, Arabinanase/levansucrase/invertase, has been modified by way of mutation for expression in Pichia pastoris. The mutations could be point mutations or deletion mutations or combinations thereof.

In an embodiment, the codon-optomized gene for P-fructofuranosidase and/or fructosyltransferase has been modified for expression in a heterologous host cell. Further, the modified gene has been fused to one or more signal peptides.

In one embodiment, the codon optimized nucleic acid encoding P-fructofuranosidase of Aspergillus niger is represented by SEQ ID NO: 6 .

In one embodiment, the codon optimized nucleic acid encoding modified fructosyltransferase of Aspergillus japonicus is represented by SEQ ID NO: 8 .

In another embodiment, the modified nucleic acid is fused to one or more signal peptide.

In another embodiment, the signal peptide is selected from Alpha-factor of S. cerevisiae (FAK), Alpha-factor full of S. cerevisiae (FAKS) of S. cerevisiae, Alpha factor_T of S. cerevisiae (AT), Alpha-amylase of Aspergillus niger (AA), Glucoamylase of Aspergillus awamori (GA), Inulinase of Kluyveromycesmaxianus (IN), Invertase of S. cerevisiae (IV), Killer protein of S. cerevisiae (KP), Lysozyme of Gallus gallus (LZ), Serum albumin of Homo sapiens (SA)

In another embodiment, the signal peptides are provided in the Table 2.

Table 2: Signal peptides

In another embodiment, the harvested P -fructofuranosidase of Aspergillus niger as well as fructosyltransferases from Aspergillus japonicus was characterized to identify the bioactive fragments. It was found that the following bioactive fragments of conserved FTases that account for the catalytic activities are provided in the Table 3 below:

Table 3: Bioactive fragments of p-fructofuranosidase are conserved and accounts for the catalytic activity.

In another embodiment, in all the secretory signal peptide sequences, a stretch of four amino acids (LEKR) was added for the efficient Kex2 processing of pre-protein provided in the Table 4. Table 4: Modified Signal Peptides used

In another embodiment, the modified nucleic acid is cloned in an expression vector.

In another embodiment, the expression vector is configured for secretory or intracellular expression of recombinant FTases from genus Aspergillus.

In yet another embodiment, the expression vector is selected from a group comprising pPICZaA, pPICZaB, pPICZaC, pGAPZaA, pGAPZaB, pGAPZaC, pPIC3, pPIC3.5, pPIC3.5K, PAO815, pPIC9, pPIC9K, pHIL-D2 and pHIL-Sl.

In another embodiment, the expression of the modified FTase gene fused to a signal peptide is preferably driven by a constitutive or inducible promoter.

In another embodiment, the nucleic acid to be expressed in operably linked to the promoter.

In another embodiment, the constitutive or inducible promoter is selected from a group listed in Table 5 below.

Table 5: List of promoters used

In another embodiment, the promoter is an AOX1 promoter, which is induced by methanol and repressed by glucose.

In an embodiment, the expression vector containing the modified gene of interest (FTase gene fused to a nucleic acid encoding signal peptide) is transformed in an appropriate host.

In an embodiment, the method of producing a recombinant host cell capable of expressing modified FTase of Aspergillus sp., process comprising the steps of: a. synthesizing a modified nucleic acid molecule from Aspergillus sp. as defined in claim 6; b. constructing a vector harboring the modified nucleic acid molecule; and c. transforming a host cell with the vector of step (b) to obtain a recombinant host cell.

In an embodiment, the codon optimized gene for P-fructofuranosidase and/or fructosyltransferase has been modified for expression in a heterologous host cell.

In an embodiment, the heterologous recombinant host cell being a prokaryotic host chosen from a group comprising Escherichia coli, Bacillus subtilis, Pseudomonas putida, Corynebacterium glutamicum and the like.

In another embodiment, the heterologous recombinant host cell being a eukaryotic host. The eukaryotic host cell can be chosen from a group comprising Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha and the like.

In another embodiment, the expression vector containing the gene of interest is transformed in yeast cells.

In another embodiment, the yeast cell is a Pichia pastoris.

In yet another embodiment, the Pichia Pastoris host cell is a mut+, mut S or mut- strains. Mut+ represents methanol utilization plus phenotype.

In yet another embodiment, the Pichia Pastoris host cell strain is selected from a group comprising KM71H, KM71, SMD1168H, SMD1168, GS115, X33. In another embodiment, the invention provides FTase pre-cursor peptides, wherein FTases of genus Aspergillus is fused to one or more signal peptide.

In an important embodiment are provided the mutants and processes of generating mutants of FTases of genus Aspergillus. Mutations can be single, double or triple point mutations as well as deletion mutants and also a combination of these mutations.

In another embodiment, are provided nucleic acid sequences of the modified P- fructofuranosidase from Aspergillus niger set forth in SEQ ID NO. 9, 11, 13, 15, 17, 19, 21,

23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 .

In another embodiment, are provided peptide sequences of the modified P- fructofuranosidase from Aspergillus niger set forth in SEQ ID NO.: 10, 12, 14, 16, 18, 20, 22,

24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 67 - 218.

In another embodiment, the signal peptide is selected from a group comprising Alpha factor full of S. cerevisiae (FAK) set forth in SEQ ID NO: 219, Alpha-factor full of S. cerevisiae (FAKS) set forth in SEQ ID NO: 229 , Alpha factor_T of S. cerevisiae (AT) set forth in SEQ ID NO: 220, Alpha-amylase of Aspergillus niger (AA) set forth in SEQ ID NO: 221, Glucoamylase of Aspergillus awamori (GA) set forth in SEQ ID NO: 222, Inulinase of Kluyveromyces maxianus (IN) set forth in SEQ ID NO: 223, Invertase of S. cerevisiae (IV) set forth in SEQ ID NO: 224 , Killer protein of S. cerevisiae (KP) set forth in SEQ ID NO: 225, Lysozyme of Gallus gallus (LZ) set forth in SEQ ID NO: 226 , Serum albumin of Homo sapiens (SA) set forth in SEQ ID NO: 227, and variants thereof.

In yet another embodiment, the mutants have been produced by conventional genetic engineering techniques as well as by utilizing bio-informatics based tools. Point mutations were predicted and introduced into the codon-optimized P-fructofuranosidase (fopA) gene of Aspergillus niger and/or ft gene of Aspergillus japonicus. Approaches such as conventional genetic engineering like PCR etc., bioinformatics tools utilizing molecular dynamics simulations using the software GROMACS as well as introduction of codon bias have been used in the present invention.

In one embodiment are provided Fructosyltransferase mutants which are presented as below:

Point mutants:

V44L, T155R, T293W, R286V, T459R, R199K, F182P

Deletion mutants:

32-654 aa, 32-194 aa, 1-194 aa In an embodiment, the process for the production of recombinant FTases from genus Aspergillus is provided.

Aspects of the present invention relate to fermentation of recombinant Pichia pastoris cells containing modified/mutated recombinant FTase gene. After completion of the fermentation, the fermentation broth is subjected to centrifugation and filtered using microfiltration and the recombinant enzyme is separated. The recovered recombinant enzyme is concentrated using Tangential Flow Ultra-filtration or evaporation and finally the concentrated enzyme is formulated.

In one embodiment, process for expressing modified FTase of Aspergillus sp. as, comprising: a. culturing recombinant host cells capable of expressing FTase of Aspergillus sp. in a suitable fermentation medium to obtain a fermentation broth; b. harvesting supernatant from the fermentation broth, wherein the supernatant contains recombinant FTase; and c. purifying recombinant FTases.

In another embodiment, the fermentation medium is basal salt medium as described in Table 6 below:

Table 6: Composition of basal salt medium In yet another embodiment, the supernatant from the fermentation broth is harvested using centrifugation.

In one embodiment, the percentage of inoculum or starter culture to initiate the fermenter culture is in the range of 2.0% to 15.0 % (v/v).

In another embodiment, the pH of the fermentation medium is maintained in the range of 4.0 to 7.5 as the secreted enzyme undergoes proper folding and is biologically active at this pH range.

In yet another embodiment, the temperature of the fermentation process is in the range of 15°C to 40°C.

In another embodiment, the time for fermentation process is in the range of 50-150 hrs.

In a further, embodiment, the fermentation broth is centrifuged at a speed in the range from 2000 xg to 15000 xg using continuous online centrifugation.

The supernatant obtained after centrifugation is subjected to microfiltration and purified to recover biologically active recombinant/mutant FTase.

In one embodiment, the supernatant obtained after centrifugation is concentrated using a Tangential Flow Filtration based Ultra filtration System.

The cut-off size of the membranes used in Tangential Flow Filtration (TFF) systems that may be used to remove impurities and to concentrate the collected culture supernatant may range between 5 to 100 kDa .

In another embodiment, no centrifugation is required for the process due to the high yield and purity of the secreted enzyme.

The FTase concentration obtained in this invention is found to be in the range of or more than 2-5 gm/L and the purity is high, say 85% or more.

In an embodiment, process to identify mutations of amino acids across a FTase protein chain including the active site, wherein the steps comprising: a. Simulating the apo structure of FTase enzyme using a bioinformatics tool; b. Clustering the trajectory based on variation of the root mean squared deviation (RMSD) to identify unique conformations; c. Identifying the unique conformations that the FTase enzyme adopted during the course of the simulation residues for mutation; d. Analyzing the identified unique conformations of step (c) for pairwise interactions that each amino acid made with another amino acid; e. Quantifying the pairwise interactions of the amino acids as an indicative interaction strength by providing appropriate weights for the van der Waals and the hydrogen bonds between the pair of residues; f. Generating 19 mutations of each amino acid that was found to have larger standard deviation in the interaction strengths through the course of the simulation; and g. Identifying and selecting stabilizing mutations with similar or better stability as per the -AAG values.

EXAMPLES

The following examples particularly describe the manner in which the invention is to be performed. But the embodiments disclosed herein do not limit the scope of the invention in any manner.

Example 1: Modified nucleic acids for expression of recombinant p-fructofuranosidase of Aspergillus niger in Pichia pastoris

The native cDNA of f> -fructofurano sidase (SEQ ID NO. 5) was codon optimized for maximizing expression in Pichia pastoris. The codon optimized nucleotide sequence of f>- fructofur ano sidase represented by (SEQ ID NO.: 6 ) is further modified by inducing mutations selected from one or more point mutations as defined in Table 7 or deletion mutations defined in 8 or their combination thereof were induced in the codon optimized nucleotide sequence. The amino acid and nucleotide sequences of modified P-fructofuranosidase is represented by SEQ ID NO.: 9 - 218 .

Table 7: A brief of the mutations in FTase proteins

Table 8: A brief of deletion mutation

An expression cassette encoding the P-fructofuranosidase was modified for maximizing expression in Pichia pastoris. The modified open reading frame contains the modified nucleotide sequence encoding P-fructofuranosidase fused to a signal peptide. The nucleic acids have been designed such that the encoded signal peptides contain an additional stretch of four amino acids (LEKR) for the efficient Kex2 processing of precursor peptide.

The preferred codons for expression in Pichia pastoris were used in place of rare codons. The nucleotide sequence of the modified open reading frames encoding for P- fructofuranosidase were fused with modified signal peptides. Recombinant pre-cursor proteins were obtained by translating the gene encoding for P-fructofuranosidase of Aspergillus niger fused with signal peptides. The signal peptides were cleaved off during post-translational modifications inside the Pichia host cells and the mature recombinant P-fructofuranosidase was released into the medium.

Example 2: Development of mutants

Example 2(a): Bio-informatics approach:

The present inventors sought to identify mutations of amino acids across the protein chain including the active site that can result in greater stabilization of the protein AnFT i.e., enzyme FT from Aspergillus niger. In order to identify such amino acids, the apo structure of AnFT was subjected to 25ns molecular dynamics simulations using the software GROMACS.

Subsequent to the simulation, the trajectory was clustered based on the variation of the root mean squared deviation (RMSD) of the CA atoms to identify unique conformations that the protein adopted during the course of the simulation. Representative structures of the clusters (cluster centres) were chosen.

These structures were analysed for the interactions that each amino acid made with another amino acid. The pairwise interactions of the amino acids were quantified as an indicative interaction strength by providing appropriate weights for the van der Waals and the hydrogen bonds between the pair of residues.

Strength of interaction between amino acids z and j, Sij = Wt/rij

Where, Wt is the weight for a given type of interaction and rij is the distance between atoms of the residues z and j. The strength of the interaction computed between two residues is the algebraic sum of the strengths of interactions between atoms of the two residues.

This yielded an NXN matrix for a protein of N residues with each element representing the interaction strength and 0 representing no interaction. Sum of each row in the matrix simply yields the overall interaction strength of a given amino acid. e.g., total interaction strength of residue 100 = sum (interaction strength of residue 100 with every other residue)

A radius of 5A was chosen as a cut off to analyse and quantify the interactions. No interaction was assumed between successive amino acids so that the interaction strength was a true reflection of the stabilization through tertiary structure. Greater interaction strength points to greater stabilization of the amino acid and hence that of the protein.

The ensemble of AnFT structures resulting from the molecular dynamics simulations followed by clustering comprised of 5-6 different conformations (cluster centres). For each of the conformation, the interaction strength matrix was generated and the individual interaction strength of each amino acid was computed as described above.

The variation of the interaction strengths of each amino acid across the ensemble was studied using the statistical parameter of standard deviation. A greater standard deviation in the interactions among the various conformations indicated a flexible residue while a lower standard deviation indicated a well networked residue. The flexible residues were targeted to generate mutants. An overview of the scheme for identification of the most stable sites for mutations is illustrated in Figure 11.

Further, each such amino acid that was found to have larger standard deviation in the interaction strengths through the course of the simulation was mutated into all other 19 natural amino acids occurring in proteins.

Furthermore, energies of stabilization were computed for the mutated amino acids with respect to the wildtype and they were represented by the parameter AAG indicating the difference between the AG for the wild type and the stabilized mutant. This analysis was carried out using the commercial software ICM from MolSoft. The AAG (kcal/mol) values for the mutation of each amino acid into 19 others were computed and those mutations which showed no further or better stabilization were selected. The list of mutations so selected are given in the Table 9 below along with the AAG (kcal/mol) values for each mutation.

Table 9

Similar exercise as described above was carried out on the kestose bound AnFT structure to analyse the strengths of interactions of the amino acids in the kestose binding site and the effect of their mutations on the stability of the protein. The results of that analysis are shown in the below Table 10 .

Table 10

As described above, utilizing a combination of molecular dynamics simulations, trajectory analyses, interactions strengths of individual amino acids and the characterization of the mutants for their stabilizing influence on the protein, a minimum of 172 sites for point mutation were identified. Further, the suitable sites for deletion were also identified based on the structural studies in the prior art on the apo structure of the FTase.

Example 2(b): Genetic engineering approach: Based on bioinformatics approaches the alternative forms of functional domains for the FTase protein were conceived and cloned to further test their efficacy for the process. For cloning work, the primers were designed to PCR amplify a particular stretch of DNA from the recombinant plasmid used as a template having codon optimized full length FTase gene. Thus, the amplified PCR products contained the required deletion and retained the desired stretch from the gene. These PCR products were cloned in the respective expression vectors with compatible strategies, for example cloned as Xhol and Sad I fragments at the corresponding sites in the MCS of vector pPICZaA.

The point mutations were introduced in the sequence of FTase gene through primers carrying the mutated nucleotides. The PCR products generated through these primers contained the mutated sequence at the corresponding site(s). These PCR products now carrying the point mutations were then cloned according to the compatible strategy in the desired expression vector.

Example 3: Development of recombinant host cells by transformation with recombinant plasmids

The vector used in the process was pPICZaA. The vectors contained the modified open reading frames and an inducible promoter, AOX1. The modified sequence encoding for the recombinant protein was cloned into the pPICZaA vector.

The modified nucleic acid encoding respective FTase gene was cloned between XhoI/SacII restriction sites present in the MCS of pPICZaA vector to bring signal sequence Alpha-factor of S. cerevisiae (FAK) in frame to create expression cassette using regular molecular biology procedures. The vector map for pPICZaA is represented in Figure 3.

The putative recombinant plasmids were selected on low salt-LB media containing 25 pg/ml Zeocin and screened by XhoI/SacII restriction digestion analysis.

The recombinant plasmid pPICZaA-FTase was confirmed by XhoVSac restriction digestion analysis which resulted in release of 1980 bp fragment. The results of the restriction digestion analysis are depicted in Figure 4.

Figure 9 depicts the results of the restriction digestion analysis performed on the recombinant plasmid pPICZaA containing (a): N32R, H43Y, H43V, H43L, V125F, V127A, P131Y; (b): F182R, F182A, F182D, F182V, F182T, T293F, T293H; (c): T293R, T293L, T293S, Q327L; (d): Q327N, V343F Thereafter, Pichia pastoris cells were electroporated with linearized recombinant pPICZaA-FTase DNA. The Pichia integrants were selected on yeast extract peptone dextrose sorbitol agar (YPDSA) containing lOOpg/ml Zeocin.

The integration was screened with colony PCR (cPCR). For cPCR, a template from each of the Pichia integrants was generated by the alkali lysis method.

The Pichia integrants were grown for 48h in BMD1 media and further induced first with BMM2 and then successively with BMM10 media which provided final concentration of 0.5% methanol in the culture medium. At the end of 96 hrs induction period, culture supernatants from different clones were harvested. Total protein from each of the harvested supernatants was precipitated with 20% TCA and analyzed on SDS-PAGE.

Upon induction FTase protein bands were seen at the size of approximately 110 kDa as depicted in Figure 5.

The calculated molecular weight was about 70.85 kDa. The increase in molecular weight may have been contributed by glycosylation.

Example 4: Fermentation of Recombinant Pichia pastoris expressing mutant FTases of Aspergillus

Fermentation of recombinant Pichia pastoris cells containing the modified FTase gene as described in Example 1 was carried out in a 50 L fermenter. Fermentation was carried out in basal salt medium as described herein.

Preparation of pre-seed and seed inoculum:

The pre-seed was generated by inoculating from the glycerol stock in 25 mL of sterile YEPG medium and growing at 30°C in a temperature-controlled orbital shaker overnight. For generating seed, the inoculum was grown in Basal salt medium in baffled shake flasks at 30°C in a temperature-controlled orbital shaker till ODeoo of 15-25 was reached.

Fermentation Process

The entire process of fermentation from the inoculation of fermenter with seed culture to final harvesting took about 130 hrs. Basal salt medium was prepared and sterilized in situ in the fermenter.

The composition of basal salt medium optimized for the fermentation process is provided in Table 6.

Pichia Trace Minerals (PTM) salt solution was prepared with the components mentioned in Table 11. PTM salts were dissolved and made up to 1 L volume and filter sterilized. PTM salt solution was included at the rate of 4ml per liter of initial media volume after sterilization of the basal salt media.

Table 11: PTM trace salts

Growth Phase:

The growth phase was initiated by inoculating basal salt medium in 50 L fermenter with 5% seed culture and continued for about 24 hours. The dissolved oxygen (DO) levels were continuously monitored and never allowed to drop below 40%.

After 18h, a DO spike was observed indicating the depletion of carbon source (Glycerol). A glycerol fed-batch was initiated by feeding 50% Glycerol (with 12 ml of PTM salts per liter of feed) for about six hours till the ODeoo reached 200.

Induction Phase:

Once sufficient biomass was generated, the induction phase was initiated by discontinuing glycerol feed and starting methanol feed. Methanol (supplemented with 12 ml of PTM salts per liter of feed) was fed at the rate of 0.5g to 3g per liter of initial fermentation volume. The DO was maintained at 40% and methanol feed was accordingly adjusted. The induction of FTase gene was monitored periodically by analyzing culture supernatant by enzyme activity assay. The induction phase was continued for about 100 hours till the ODeoo reached 600 and wet biomass reached -560 grams per liter of culture broth.

The fermentation was stopped after 130 hours and enzyme activity in the fermenter broth was determined at the end of fermentation by DNS method (Miller, 1959). One unit is defined as the amount of enzyme required to release one micromole of reducing sugars (glucose equivalents) from 10% sucrose solution in 100 mM citrate buffer pH 5.5 at 55°C. The total amount of recombinant FTase in the culture broth was estimated by Bradford assay. Fermentation conditions:

The fermentation parameters considered were as given in Table 12. These essential parameters were monitored during the fermentation process.

Table 12: Fermentation Parameters

Example 5: Cell Harvesting and purification

Harvesting of the enzyme was performed by continuous centrifugation at 8000 RPM. Clear supernatant obtained after centrifugation was subjected to microfiltration using 0.1 microns cut off spiral wound TFF membrane. The filtrate was further subjected to ultrafiltration and diafiltration using 10 kDa cutoff spiral wound TFF membrane and sufficiently concentrated and to reach the desired activity. The enzyme was formulated by including 35- 50% of glycerol and food-grade preservatives in the final preparation. The final purity of the enzyme was observed to be 85% as determined by SDS -PAGE analysis.

SDS-PAGE analysis of samples was collected at different time intervals during fermentation of Pichia pastoris strain expressing recombinant FTase enzyme was also performed. SDS-PAGE analysis of recombinant Ftase enzyme after purification was also performed. Figure 5 depicts the SDS-PAGE analysis for screening protein expression in Pichia pastoris recombinant strains having integration (a): pPICZaA-V44L; (b): pPICZaA-T155R; (c): pPICZaA-T293W; (d): pPICZaA-R286V; ©: pPICZaA-R199K; (f): pPICZaA-T459R; (g): pPICZaA-F182P.

Figure 6 depicts the SDS-PAGE analysis of samples collected at different time intervals during fermentation trail of various mutant Ftase proteins, (a): pPICZaA-V44L; (b): pPICZaA- T155R; (c): pPICZaA-T293W; (d): pPICZaA-R286V; ©: pPICZaA-R199K; (f): pPICZaA- T459R; (g): pPICZaA-F182P

Figure 10 depicts the SDS-PAGE analysis for screening protein expression in Pichia pastoris recombinant strains having integration (a): pPICZaA-H43Y; (b): pPICZaA-H43L; (c): pPICZaA-Q327N; (d): pPICZaA-V343F

The Ftase concentration was found to be about 2.4 gm/L. In most of the batches, the concentration was 2-5 gm/L. The purity of the recombinant Ftase was observed to be about 85%.

Example 6: Estimation of Ftase activity

Studies were conducted to estimate the activity of Ftase. For the estimation studies, the amount of reducing sugar generated due to the action of Ftase enzyme was calculated using DNS (3,5 Dinitrosalicylic acid) method (G. L. Miller, “Use of dinitrosalicylic acid reagent for determination of reducing sugar”, Anal. Chem., 1959, 31, 426-428).

For conducting the enzyme activity assay, 10% Sucrose (dissolved in lOOmM Citrate buffer) was used as the substrate. Ftase was recovered from the fermentation broth and processed through ultra-filtration. The ultra-filtered sample was then diluted 25,000X by serial dilution in lOOmM Citrate buffer and was used. The reaction volume was 2.5mL. The pH was maintained at 5.5 and the reaction was continued for 15 minutes.

After incubation 3 mL of DNS (3,5 Dinitrosalicylic acid) was added to each reaction mixture and boiled for 10 min, cooled and read absorbance at 540 nm, spectrophotometrically. The OD of glucose at different concentration was measured as shown in Table 13. Thereafter, based on the absorbance measurement after the reaction, the enzyme activity was calculated as shown in Table 14.

Table 13: OD measurement of glucose at different concentration

Table 14: Estimation of activity of FTase

The FTase activity of different clones were estimated after 96 hrs of methanol induction. Supernatant of each clone was subjected to TCA precipitation. The TCA prepped sample was then tested for FTase activity (by DNS method) according to the reference mentioned above. Multiple clones of each construct were tested for FTase activity. The data from representative clones have been presented in Table 15. The FTase activity of the wild type molecule found to be 290 Units/ml was considered as experimental control.

The FTase activity of mutant variants namely but not limited to N32R, H43V, V 125F, V 127A, P131Y, T155R, F182P, F182R, F182A, F182D, F182V, F182T, R286V, T293F, T293H, T293L, T293S, T293W, H43Y, H43L, Q327N, Q327L and V343F were found to be surprisingly well.

Table 15

Example 7: Generation of fructooligosaccharides (EOS) from Sucrose and recombinant FTase enzyme

Studies were conducted to understand the ability of the enzyme in the formation of fructooligosaccharides. A 100 mL solution of 90% (w/v) Sucrose was prepared in 150 mM sodium citrate buffer pH 5.5. To this, 96.7 pL of FTase enzyme having 51692 Unit/ml of activity (equivalent to total of 5000 Units of enzyme), was added.

The reaction was set up in a 250 mL conical flask and incubated at 65°C and 220 rpm. At regular time intervals, samples were taken and analyzed on Thin Layer Chromatographic (TLC) plates.

Glucose, sucrose, fructose and FOS (containing kestose, nystose and fructofuranosylnystose) were used as standards for the thin layer chromatographic analysis. The mobile phase used was n-Butanol: Glacial acetic acid: Water (4:2:2 v/v) and the developing / staining solution used was urea phosphoric acid.

Figure 7 depicts the TLC analysis done for the generation of fructooligosaccharides (FOS) from sucrose and recombinant FTase enzyme.

The sample was further subjected to High Performance Liquid Chromatography (HPLC) for quantitative estimation of the production of fructooligosaccharides. The HPLC analysis was done using an amine column (Zorbax NH2 column, Agilent Technologies) having 4.6 (ID) x 150 mm (length) and 5 pm (particle size). The standard solutions of glucose, fructose, kestose, nystose, fructofuranosylnystose and sucrose of different concentrations were run for generating standard curves.

Figure 8 depicts the HPLC analysis chromatogram of FOS samples.

Example 8: Characterization of recombinant FTase of Aspergillus sp.

The harvested FTase of Aspergillus sp. selected from but not limited to fructosyltransferase of Aspergillus japonicus , P-fructofuranosidase of Aspergillus niger was characterized to identify bioactive fragments. It was found that following bioactive fragments of are conserved and accounts for the catalytic activities as illustrated in Table 3 above.

Similar approach as described above was followed for creating mutants of FTase of Aspergillus japonicus. The protein sequence of FTase in A. niger and A. japonicus differ only at four loci out of 654 loci namely, 44, 199, 459 and 654 as illustrated in Figure 12. In terms of activity, both the enzymes (from genes ft ad fopA ) were found to be comparative with each other.

ADVANTAGES:

1. The enzymes exhibits high purity after filtration, which eliminates the need for costly chromatographic procedures.

2. The enzymes can be used for obtaining a high yield of fructooligosaccharides.