The present invention relates to a process for converting a pentose into 1 ,2,4-butanetriol, a composition for converting a pentose into 1 ,2,4-butanetriol, an aqueous composition and the use of a lactonase in the conversion of a pentose into 1 ,2,4-butantetrioL

1 ,2,4-Butanetriol (BTO) is a versatile chemical with various applications. On the commercial level (R-) and (S-)1 ,2,4-butanetriol have previously been obtained by high-pressure catalytic hydrogenation of D,L-malic acid. The reaction is based on the reduction of esterified D,L-malic acid with NaBF under high pressure. Such synthesis techniques produce a large number of by-products, and for every ton of 1 ,2,4-butanetriol synthesized, several tons of by-products are produced. In addition to that, current substitutes for the high-pressure catalytic hydrogenation of D,L-malic acid used to obtain 1 ,2,4-butanetriol are expensive, have low yields or are generally impractical for large-scale use.

Currently, 1 ,2,4-butanetriol is in particular of great interest in the military sector as a feedstock for providing 1 ,2,4-butanetriol trinitrate, which is used as a propellant for military weapons such as aircrafts, missiles, guided missiles etc. It has several advantages over nitroglycerin. In particular it is less sensitive to handle, has improved thermostability and lower volatility. This makes it a much safer alternative. Nitroglycerin consumption in the United States for dual-fuel propellants currently exceeds 1 ,300,000 tons/year when fully replaced by 1 ,2,4-butanetriol trinitrate; the market for 1 ,2,4-butanetriol trinitrate for the U.S. military is at least 1 ,700,000 tons/year.

Apart of that, 1 ,2,4-butanetriol can also be used for the production of biologically active agents, pharmaceutical sustained release, cigarette additives, antiseptic germicidal agents, color developers etc.

Therefore, biocatalytic routes for the production of 1 ,2,4-butanetriol have been increasingly considered.

This was first demonstrated by US 7923226 B2. However, subsequent work showed that a fermentative process using an adapted microorganism still has a great many problems. The required enzyme activities are not all native. Very large differences in the individual enzyme activities lead to the accumulation of intermediates especially of xylonic acid and its salts. This leads to a shift of the pH value as well as to a strong imbalance of the cofactor balance in the cell.

For above reasons, improved processes for the preparation of 1 ,2,4-butanetriol are necessary. It would be desirable for the process to provide good yields of 1 ,2,4-butanetriol combined with complete substrate conversion and be suitable for commercial and large-scale applications. Besides, minimizing the creation of by-products would also be desirable. Allowing a process with a high concentration of 1 ,2,4-butanetriol to reduce the afford for down-streaming.

It is the object of the present invention to provide a process for the production of 1 ,2,4- butanetriol providing 1 ,2,4-butanetriol in high yields. Minimizing the amount and yield of byproducts is also an object. Besides, a process being suitable for commercial and large-scale applications is also desirable.

This object is solved by the process for converting a pentose into 1 ,2,4-butanetriol according to the present invention, the composition for converting a pentose into 1 ,2,4-butanetriol, the aqueous composition and 1 ,2,4-butanetriol obtainable by the process according to the present invention and the use of a lactonase in the conversion of a pentose into 1 ,2,4-butantetriol according to the invention.

In a first aspect the invention relates to a process for converting a pentose into 1 ,2,4-butanetriol comprising, preferably consisting of, the steps of: a) adding to a composition comprising water, at least one co-factor and a pentose, at least five enzymes, and b) subsequently enzymatically converting the pentose to 1 ,2,4-butanetriol in the presence of the at least five enzymes, wherein in step a) the at least five enzymes are selected from the group consisting of dehydrogenase, dehydratase, lactonase, decarboxylase and combinations thereof, and wherein at least one enzyme in step a) is a lactonase.

It has been surprisingly found that the inventive process achieves the enzymatic production of 1 ,2,4-butanetriol starting from a pentose in high yields. Besides, the inventive process tolerates high concentration of substrate as well as product.

The present invention provides a process in particular a biocatalytic process, synthesis methods, materials and organisms for preparing the enzymes for providing 1 ,2,4-butanetriol from a carbon source. The classification or discussion of a material in any section of this specification as having a particular utility (e.g., "catalyst") is for convenience.

The citation of references herein does not constitute an admission that such references are prior art or relevant to the patentability of the invention disclosed herein. Any discussion of the contents of the references cited is intended to provide only a general summary of the claims made by the authors of the references and does not constitute an admission as to the correctness of the contents of such references.

While the description and specific examples provide embodiments of the invention, they are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, mention of several embodiments having the features mentioned is not intended to exclude other embodiments having additional features or other embodiments having other combinations of the features mentioned. Specific examples are provided to illustrate how the compositions and methods of the present invention may be made and used and, unless otherwise expressly stated, are not to be construed as representations that particular embodiments of the present invention have or have not been made or tested.

As used herein, the words "preferred" and "preferably" refer to embodiments of the invention that provide certain advantages under certain circumstances. However, other embodiments may also be preferred under the same or different circumstances. Moreover, mention of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

Reference to pentose in the context of the invention is to be understood as reference to D- pentose and L-pentose.

Reference to 1 ,2,4-butanetriol in the context of the invention is to be understood as reference to (R)-1 ,2,4-butanetriol and (S)-1 ,2,4-butanetriol.

The bioconversion processes of the present invention are based on de novo creation of biosynthetic pathways wherein (R)-1 ,2,4-butanetriol and/or (S)-1 ,2,4-butanetriol are synthesized from a carbon source.

The advantage of the inventive process is that the use of five enzymes wherein one enzyme is a lactonase accelerates the process and increases the overall yield of the inventive process. The lactonase catalyzes the carboxylic ester hydrolysis of hexono-1 ,5-lactones as well as pentono-1 ,5-lactones, also referred to as the ring-opening.

According to a preferred embodiment the pentose can be a mixture of different pentoses. In another preferred embodiment the pentose is an aldopentose.

It is preferred that the pentose is selected from ribose, arabinose, xylose and/or lyxose, preferably the pentose is selected from D-ribose, D-arabinose, L-arabinose, D-xylose and/or D-lyxose, more preferably is selected from L-arabinose or D-xylose and most preferably is D- xylose. The process also allows the use of different pentoses, that is, mixtures of aforementioned pentoses.

It is preferred that the pentose is L-arabinose and/or D-xylose. D-xylose and L-arabinose are the predominant carbohydrates from corn fiber and sugar beet pulp (Salnier, L.; Marot, C.; Chanliaud, E.; Thibault, J.-F. Carbohydr. Polym. 1995, 26, 379. Micard, V.; Renard, C. M. G. C.; Thibault, J.-F. Enzyme Microb. Technol. 1996, 19, 162.) The opposite stereogenic C-4 centers of D-xylose and L-arabinose, which form the basis for the synthesis of (R)-1 ,2,4- butanetriol and (S)-1 ,2,4-butanetrioL

Preferably the lactonase is present in an amount of from 0.01 to 20 pM and more preferably from 0.1 to 10 pM.

In the process a co-factor is used. Every suitable co-factor known to the person skilled in the art can be applied. Preferably a redox co-factor is used in the inventive process.

It is preferred that the co-factor applied in step a) is NAD ⁺ and/or NADH/H ⁺ .

Preferably the co-factor is recycled in the process.

This leads to a significant reduction in the co-factor input. One of the positive effects is the reduction of the costs of the co-factor as well as a reduction in substances to be added to the process leading to additional impurities.

According to a preferred embodiment in step b) of the inventive process the enzymatical conversion is a one-pot reaction.

An advantage of the inventive process is that it performs all the catalytic steps in the same reaction batch without the need to isolate intermediates neither to add enzymes or substrates for cofactor recycling. The process can be operated either batchwise or continuously.

The enzymes present in the reaction mixture can be removed, for example, physically (filtration or via immobilization) or inactivated. The latter can be done by a short-term increase in temperature to, for example, 80°C for 10 min. Alternatively, the reaction solution can also run through the reactor containing the enzymes for increased conversion and yields several times.

It is preferred that the concentration of the pentose in step a) is between 10 and 500 g/L, preferably between 50 and 450 g/L, more preferably between 100 and 300 g/L, even more preferably 120 to 280 g/L, and even more preferably between 140 to 250 g/L.

In general, high substrate load is always preferred in industry. At high concentrations, microbial contamination issues can be minimized. However, the problem is regularly that in case the concentration of initial pentose is increased to a level which produces BTO at the toxicity limit of cells, i.e. 200 g/L (1.9 M), high loads of pentose are not useful. Besides, high amounts of 1 ,2,4-butanetriol can inhibit or decrease the activity of enzymes in the cascade. Besides, high amounts of pentose and side products produced during the reaction can also have an inhibitory effect on the enzymes of the cascade.

Preferably a sixth enzymes is added in step a). More preferably this sixth enzyme has a side product of the cascade reaction as substrate. Even more preferably this enzyme is a carbonic anhydrase.

It is further preferred that the overall yield of 1 ,2,4-butanetriol obtained step b) is between 90 and 99.5% based on the overall amount of pentose added in step a).

According another preferred embodiment in step a) at least one dehydrogenase, preferably a dehydrogenase derived from Herbaspirillum seropedica and/or Dickey dadantii, more preferably a dehydrogenase derived from Herbaspirillum seropedicae with a sequence according to SEQ ID NO: 1 or SEQ ID NO: 3 or derived from Dickey dadantii with a sequence according to SEQ ID NO: 5 is added; and/or in step a) a lactonase derived from Noviherbaspirillum massiliense, preferably a lactonase derived from Noviherbaspirillum massiliense with a sequence according to SEQ ID NO: 7 is added; and/or in step a) at least one dehydratase, preferably a dehydratase derived from Paralcaligenes ureilyticus, Fontimonas thermophila, Herbaspirillum seropedicae and/or Caulobacter vibrioides, more preferably a dehydratase derived from Paralcaligenes ureilyticus with a sequence according to SEQ ID NO: 9, Fontimonas thermophila with a sequence according to SEQ ID NO: 11 , Herbaspirillum seropedicae with a sequence according to SEQ ID NO: 13 and/or Caulobacter vibrioides with a sequence according to SEQ ID NO: 15 is added; and/or in step a) at least one decarboxylase, preferably a decarboxylase derived from Lactococcus lactis, more preferably a decarboxylase derived from Lactococcus lactis with a sequence according to SEQ ID NO: 17 is added; and/or in step a) at least one dehydrogenase, preferably a dehydrogenase derived from Escherichia coli, more preferably a variation of AdhZ3 and even more preferably AdhZ3-LND according to SEQ ID NO:19, is added; and/or in step a) at least one decarboxylase, preferably a branched-chain decarboxylase is added.

Preferably at least 50 % and more preferably 70 % of the pentose present in the composition of step a) is converted to 1 ,2,4-butanetriol after 24 h of enzymatical conversion.

Further preferred at least 90 % of the pentose present in the composition of step a) is converted to 1 ,2,4-butanetriol after 48 h of enzymatical conversion.

According to a preferred embodiment the Space-Time-Yield (STY) is at least 1.0 g/L/h, preferably 2.0 g/L/h and more preferably 2.5 g/L/h. It is preferred that the STY is between 2.0 and 25 g/L/h.

The Space-Time-Yield (STY) is defined as the volumetric productivity of BTO production.

Preferably the inventive process is carried out at suitable temperatures, e.g. may depend on the enzymes used. Suitable temperatures include 10 to 100 °C, preferably 10 to 90 °C, more preferably 20 to 90 °C, even more preferably 20 to 80 °C.

Preferably the temperature in step b) is between 10 - 100 °C, more preferably between 20 - 90 °C and even more preferably between 20 - 80 °C.

It is preferred that the pH of the composition in the inventive process is between 3 to 12, and preferably 4 to 10.

It is also possible to supplement the aqueous system by a buffer system. Suitable buffers (systems) are known and include conventional buffers (systems), for example, acetate, potassium phosphate, Tris-HCI, glycylglycine and glycine buffers, or mixtures of these.

Preferably, a buffer used in a method of the present invention has a pH of 3 to 12, preferably 4 to 12, more preferably 4 to 11 . For optimum activity of the biocatalysts ions, e.g. Mg ²⁺ , may be added. The use of stabilizers, glycerol etc. may allow longer use of biocatalysts.

Further preferred the pH of the composition is between 3 to 12 and more preferably 4 to 10. According to a preferred embodiment the inventive process comprises, preferably consists of, the following steps in the following order

(i) oxidation using a dehydrogenase, preferably xylose dehydrogenase, and the oxidized form of a co-factor, preferably NAD+;

(ii) hydrolysis to obtain a first intermediate, preferably xylonic acid, arabinonic acid, arabinonic acid, ribonic acid or mixtures thereof;

(iii) dehydration using a dehydratase to obtain a second intermediate, preferably 2-keto-3- deoxy-xylonic acid, 2-keto-3-deoxy-arabinonic acid or mixtures thereof;

(iv) decarboxylation using a decarboxylase to obtain a third intermediate, preferably 3,4- dihydroxybutanal; and

(v) reduction using a dehydrogenase and the reduced form a co-factor, preferably NADH, to obtain 1 ,2,4-butanetrioL

It is preferred that in step (ii) a lactonase is used.

Preferably in step (ii) a lactonase in an amount of from 0.01 to 20 pM and more preferably from 0.1 to 10 pM is used.

It is further preferred that at least one of the intermediates, preferably the first intermediate is added to the process.

It is to be understood and known by the person skilled in the art that the obtained intermediates also cover the respective salts.

Adding an intermediate has the effect that the Space-Time Yield (STY) is increased while the amount of co-factor can be reduced. When considering the price of cofactor and intermediate, this effect of adding an intermediate is even more impressive. For example, NAD ⁺ has a list price of about 20 € per g, while e.g. D-xylonate is estimated to cost about 1.2 € per g (the actual list price is unavailable and calculated as 10-fold higher than pure D-xylose). As cofactor price is one of the most contributing costs for in vitro enzymatic biotransformation this effect is advantageous. In particular, when considering that reducing the concentration of cofactor used will intuitively result in a slower STY due to lower intermediate concentration available to drive the reaction forward.

According to a preferred embodiment the amount of co-factor in the composition is between 0.01 to 10 mM, preferably 0.05 to 5 mM, more preferably between 0.08 to 3 mM and even more preferably between 0.1 and 2 mM. Further preferred at least one of the intermediates, preferably the first intermediate, is added in an amount of from 0.1 to 50 mM, preferably from 1 to 30 mM, and more preferably 2 to 30 mM to the process and/or the amount of co-factor in the composition is between 0.1 and 2 mM.

The present invention provides processes for the preparation of 1 ,2,4-butanetriol which allow the conversion of pentose (D-xylose, L-arabinose, D-arabinose, D-ribose or D-lyxose) or mixtures. These processes are based on a combination of enzymes. This makes it possible to completely circumvent the current disadvantages of fermentative production processes.

The present invention further provides: enzymes which have the various activities required for

A. oxidation of

(a) D-xylose to D-xylono-1 ,5-lactone;

(b) L-arabinose to L-arabino-1 ,5-lactone;

(c) D-arabinose to D-arabino-1 ,5-lactone;

(d) D-ribose to D-ribino- 1 ,5-lactone;

(e) D-lyxose to D-lyxo-1 ,5-lactone;

B. ring opening of

(a) D-xylono-1 ,5-lactone to D-xylonic acid;

(b) L-arabino-1 ,5-lactone to L-arabinonic acid;

(c) D-arabino-1 ,5-lactone to D-arabinonic acid;

(d) D-ribono-1 ,5-lactone to D-ribonic acid;

(e) D-lyxo-1 ,5-lactone to D-lyxonic acid;

C. dehydration of

(a) D-xylonic acid to 2-keto-3-deoxy-D-xylonic acid;

(b) L-arabinonic acid to 2-keto-3-deoxy-L-arabinonic acid;

(c) D-arabinonic acid to 2-keto-3-deoxy-D-xylonic acid;

(d) D-ribonic acid to 2-keto-3-deoxy-D-xylonic acid;

(e) D-lyxonic acid to 2-keto-3-deoxy-D-xylonic acid;

D. decarboxylation of 2-keto-3-deoxy-D-xylonic acid/2-keto-3-deoxy-L-arabinonic acid to (R)-3,4-dihydroxy-butanal/(S)-3,4-dihydroxy-butanal; E. reduction of (R)-3,4-dihydroxy-butanal/(S)-3,4-dihydroxy-butanal to (R)-1 ,2,4- butanetriol/(S)-1 ,2,4-butanetriol.

According to a preferred embodiment the inventive process is cell-free.

In a second aspect the invention relates to a composition for converting a pentose into 1 ,2,4- butanetriol comprising, preferably consisting of,

- water;

- at least one co-factor, preferably NAD ⁺ or NADH/H ⁺ ;

- at least five enzymes, wherein the at least five enzymes are selected from the group consisting of dehydrogenase, dehydratase, lactonase, decarboxylase and combinations thereof, wherein at least one enzyme is a lactonase; and

- pentose.

All remarks with respect to the process for converting a process for converting a pentose into 1 ,2,4-butanetriol, in particular with respect to the enzymes, as mentioned hereinbefore also apply to the composition for converting a pentose into 1 ,2,4-butanetriol where applicable.

A third aspect of the invention relates to an aqueous composition comprising, preferably consisting of, at least 0.1 mol, preferably at least 0.25 mol, more preferably between 0.5 to 2.00 mol and even more preferably 0.8 to 1.5 mol of 1 ,2,4-butanetriol; less than 0.5 mol, preferably less than 0.25 mol, more preferably between 0.001 and 0.25 mol and even more preferably between 0.01 to 0.1 mol of pentose, preferably D- xylose, L-arabinose or a mixture thereof, more preferably D-xylose or a mixture of D- xylose and L-arabinose; co-factor, preferably NADH; and less than 50 mg/mL, preferably less than 30 mg/mL, more preferably between 0.01 to 30 mg/mL, and even more preferably between 0.1 to 20mg/mL of at least five enzymes, wherein one enzyme is a lactonase, wherein the at least five enzymes are selected from the group consisting of dehydrogenase, dehydratase, lactonase, decarboxylase and combinations thereof.

All remarks with respect to the process for converting a pentose into 1 ,2,4-butanetriol, in particular with respect to the enzymes, and to the composition for converting a pentose into 1 ,2,4-butanetriol also apply to this aspect where applicable.

A fourth aspect of the present invention relates to an aqueous composition according to the invention obtainable by the inventive process.

All remarks with respect to the process for converting a pentose into 1 ,2,4-butanetriol, in particular with respect to the enzymes, the composition for converting a pentose into 1 ,2,4- butanetriol and the aqueous composition also apply to this aspect where applicable.

A fifth aspect relates to 1 ,2,4-butanetriol, preferably (R)-1 ,2,4-butanetriol and/or (S)-1 ,2,4- butanetriol, obtainable by the inventive process.

All remarks with respect to the process for converting a pentose into 1 ,2,4-butanetriol, in particular with respect to the enzymes, the composition for converting a pentose into 1 ,2,4- butanetriol and the aqueous composition also apply to this aspect where applicable.

A sixth aspect of the present invention relates to the use of a lactonase, preferably a lactonase derived from Noviherbaspirillum massiliense, in the conversion of a pentose into 1 ,2,4- butanetriol.

All remarks with respect to the process for converting a pentose into 1 ,2,4-butanetriol, in particular with respect to the enzymes, the composition for converting a pentose into 1 ,2,4- butanetriol and the aqueous composition also apply to this aspect where applicable.

Brief description of the figures

Fig. 1 Schematic representation of the enzymatic biotransformation of D-xylose to (S)- 1 ,2,4-butanetriol; Fig. 2 the effect of addition A/mLacll (Lac) (SEQ ID NO: 7) on the rate of biotransformation of D-xylose to (S)-BTO;

Fig. 3 the influence of NAD ⁺ and D-xylonate on the Space-Time Yield (STY) of the biotransformation of D-xylose to (S)-BTO. In all cases, approximately 180 g/L D- xylose was used.

Fig. 4 A) Effect of NAD ⁺ concentration (0.066g/L to 3.3 g/L) on conversion to (S)-BTO; B) combined effect of NAD ⁺ concentration (0.066g/L to 3.3 g/L) and D-xylonate addition (4.1 g/L) on conversion to (S)-BTO.

Fig. 1 shows the schematic representation of the enzymatic biotransformation of D-xylose to (S)-1 ,2,4-butanetrioL The pathway consists of five enzymatic processes with pH and redox neutral (Fig. 1 ). The process comprises the step of an NAD ⁺ assisted enzymatic oxidation of the pentose to a lactone, in Fig. 1 a D-xylonolactone. This is catalyzed by a dehydrogenase; in the process according to Fig. 1 HsXylDHI (SEQ ID NO: 1 ) is applied. This is followed by a hydrolysis, ring-opening, using a lactonase, namely A/mLacll (SEQ ID NO: 7), to obtain an acid or its respective salt; in the biotransformation shown in Fig. 1 D-xylonate is obtained. This is followed by a dehydration using a dehydratase (PuDHT (SEQ ID NO: 9)) to obtain 2-keto-3- deoxy-D-xylonate as shown in Fig. 1. In the next step is a decarboxylation catalyzed by a decarboxylase, here L/KdcA (SEQ ID NO: 17) to obtain (s)-3,4-dihydroxybutanal. In the final step a NADH assisted reduction is performed to obtain (S)-1 ,2,4-butanetriol using a dehydrogenase being in the process shown in Fig. 1 EcAdhZ3-LND (SEQ ID NO: 19). The cell-free production of BTO as shown in Fig. 1 is considered a viable alternative to fermentation as issues such as toxicity to cells is avoided.

While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiments, but that the invention will include all embodiments falling within the scope of the appended claims. EXPERIMENTAL SECTION

Materials and Methods

D-Xylose, L-Arabinose, D-Arabinose, D-Ribose, D-Lyxose were obtained commercially.

Enzyme production and purification

In brief, a plasmid encoding respective enzyme was used to transform E. coli BL21 (DE3).

PuDHT (SEQ ID NO: 9) was expressed in TB media and induced using IPTG as described previously (Sutiono. S.; Teshima. M.; Beer. B.; Schenk. G.; Sieber. V. Enabling the Direct Enzymatic Dehydration of D-Glycerate to Pyruvate as the Key Step in Synthetic Enzyme Cascades Used in the Cell-Free Production of Fine Chemicals. ACS Catal. 2020. 10 (5). 31 IQ- 3118. https://doi.org/10.1021/acscatal.9b05068).

EcAdhZ3-LND (SEQ ID NO: 19) was expressed in autoinduction media containing 0.1 mM ZnCh at 37 °C for 3 h. before shifting the temperature to 16 °C for 16 h (Pick. A.; Ruhmann. B.; Schmid. J.; Sieber. V. Novel CAD-like Enzymes from Escherichia Coli K-12 as Additional Tools in Chemical Production. Appl. Microbiol. Biotechnol. 2013. 97 (13). 5815-5824. https://doi.org/10.1007/s00253-012-4474-5; Pick, A., Ott, W., Howe, T., Schmid, J., & Sieber, V.. Improving the NADH-cofactor specificity of the highly active AdhZ3 and AdhZ2 from Escherichia coli K-12. Journal of Biotechnology, 2014 189, 157-165. https://doi.Org/10.1016/j.jbiotec.2014.06.015). Cell pellet was harvested and kept at -80 °C prior to purification.

HsXylDHI (SEQ ID NO: 1 ), HsXylDHII (SEQ ID NO: 3) and A/mLacll (SEQ ID NO: 7) were expressed in autoinduction media at 37 °C for 3 h. before shifting the temperature to 16 °C for 16 h (Sutiono, S., Pick, A., & Sieber, V. (2021 ). Converging conversion-using promiscuous biocatalysts for the cell-free synthesis of chemicals from heterogeneous biomass. Green Chemistry 2021. 23 (10), 3656-3663. https://doi.org/10.1039/D0GC04288A). Cell pellet was harvested and kept at -80 °C prior to purification.

F/DHT (SEQ ID NO: 11) was expressed in TB media and induced using IPTG as described previously (Sutiono. S.; Teshima. M.; Beer. B.; Schenk. G.; Sieber. V. Enabling the Direct Enzymatic Dehydration of D-Glycerate to Pyruvate as the Key Step in Synthetic Enzyme Cascades Used in the Cell-Free Production of Fine Chemicals. ACS Catal. 2020. 70 (5). 31 IQ- 3118. https://doi.org/10.1021/acscatal.9b05068).

HsAltDHT (SEQ ID NO: 13) and CcManDHT (SEQ ID NO: 15) were expressed in TB media and induced using IPTG. A preculture was grown at 25 ml at 30 °C overnight with 150 rpm. TB media 1000 ml in 5 L baffled flask supplemented with kanamycin was inoculated with 10 ml preculture and incubated at 37 °C with 95 rpm until ODeoo reached 0.8 to 1 . IPTG was added to a final concentration of 0.5 mM and the culture was incubated for 16 h at 20 °C. Cell pellet was harvested and kept at -80 °C prior to purification.

L/KdcA (SEQ ID NO: 17) was expressed in autoinduction media at 37 °C for 3 h. before shifting the temperature to 25 °C for 16 h (Sutiono, S., Satzinger, K., Pick, A., Carsten, J., & Sieber, V. To beat the heat-engineering of the most thermostable pyruvate decarboxylase to date. RSC advances, 2019 9(51 ), 29743-29746. https://doi.org/10.1039/C9RA06251 C). Cell pellet was harvested and kept at -80 °C prior to purification.

DdFucDH (SEQ ID NO: 5) was expressed in autoinduction media containing at 37 °C for 3 h. before shifting the temperature to 16 °C for 16 h. Cell pellet was harvested and kept at -80 °C prior to purification.

All enzymes expressed harboring hexahistidine in their N-terminal.

For purification, the cell pellet was first disrupted using sonicator at 80% and 0.5 s cycle. The cell debris was cleared out by means of centrifugation. All enzymes were then purified using Akta purifier using His-Trap column FF Crude 5 mL (GE Healthcares. Germany). The buffer was then changed to 50 mM HEPES pH 7.5 using HiPrep desalting column 26/10 50mL (GE Healthcare. Germany). All enzyme was flash frozen in liquid N2 and prior to storage at -80°C until further use.

Synthesis of intermediates

D-Xylonate, D-arabinonate, L-arabinonate, D-ribonate, D-lyxonate were produced after oxidation using gold catalyst (Sperl, J. M., Carsten, J. M., Guterl, J. K., Lommes, P., & Sieber, V. Reaction design for the compartmented combination of heterogeneous and enzyme catalysis. ACS Catalysis, 2016 6(10), 6329-6334. https://doi.org/10.1021/acscatal.6b01276). 2-Keto-3-deoxy-D-xylonate (KDX) and 2-keto-3-deoxy-L-arabinonate (KDA) were produced from D-xylonate and L-arabinonate using PuDHT (SEQ ID NO: 9). D-Xylonate, L-arabinonate, KDX and KDA were quantified using HPLC as described previously (Sutiono. S.; Siebers. B.; Sieber. V. Characterization of Highly Active 2-Keto-3-Deoxy-L-Arabinonate and 2-Keto-3- Deoxy-D-Xylonate Dehydratases in Terms of the Biotransformation of Hemicellulose Sugars to Chemicals. Appl. Microbiol. Biotechnol. 2020. 104. 7023-7035). (S)-Dihydroxybutanal (DHB) was produced from KDX after incubating with L/KdcA in 250 mM HEPES. pH 7.25 containing 0.1 mM thiamine diphosphate (TDP) and 5 mM MgCh. (R)-Dihydroxybutanal (DHB) was produced from KDA after incubating with L/KdcA (SEQ ID NO: 17) in 250 mM HEPES. pH 7.25 containing 0.1 mM thiamine diphosphate (TDP) and 5 mM MgCI?. The production of DHB was followed using HPLC measuring the decrease of KDX or KDA. After 4 h. no more KDX or KDA peak was observed and a single peak in the same retention time as 1 ,2,4-butanetriol (BTO) was detected, thus >99% yield for DHB production was assumed.

Biotransformation of D-xylose to (S)-1 ,2,4-butanetriol

The cell-free bioproduction of 1 ,2,4-butanetriol (BTO) was performed in 500 pl scale in Eppendorf tube. The solution contained the combination of enzymes as shown in Table 1 . TDP 0.1 mM. 5 mM MgCh. and 50 mM HEPES pH 7.5. and 1.25 M D-xylose. The reaction was carried out in triplicates. The formation of BTO was followed over time by withdrawing 20 pl of aliquot at certain time intervals. The sample was diluted 25-fold using 5 mM H2SO4 prior to filtration through 10 KDa spin column (VWR. Germany). The filtrate was analyzed by HPLC using an ion-exclusion column (RezexROA-Organic Acid H+(8%. Phenomenex. Germany) run isocratically using 2.5 mM H2SO4 at 70 °C for 20 min.

Table 1 : Enzyme amounts for biotransformation of D-xylose to (S)-BTO

*STY or space-time yield is defined as the volumetric productivity of (S)-BTO production. SP or specific productivity is defined as the amount of (S)-BTO formed per hour per g of total biocatalyst used.

Biotransformation of L-arabinose to (R)-1 ,2,4-butanetriol

The cell-free bioproduction of BTO was performed in 500 pl scale in Eppendorf tube. The solution contained combination of enzymes according to Table 2. TDP 0.1 mM. 5 mM MgCh. and 50 mM HEPES pH 7.5. and 1 .25 M L-arabinose. The reaction was carried out in triplicates. The formation of BTO was followed over time by withdrawing 20 pl of aliquot at certain time intervals. The sample was diluted 25-fold using 5 mM H2SO4 prior to filtration through 10 KDa spin column (VWR. Germany). The filtrate was analyzed by HPLC using an ion-exclusion column (RezexROA-Organic Acid H+(8%. Phenomenex. Germany) run isocratically using 2.5 mM H2SO4 at 70 °C for 20 min. Table 2: Enzyme Amounts for biotransformation of L-arabinose to (R)-BTO

*STY or space-time yield is defined as the volumetric productivity of (S)-BTO production. SP or specific productivity is defined as the amount of (S)-BTO formed per hour per g of total biocatalyst used.

Biotransformation of D-arabinose to (S)-1,2,4-butanetriol

The cell-free bioproduction of BTO was performed in 500 pl scale in Eppendorf tube. The solution contained combination of enzymes as shown in Table 3. TDP 0.1 mM. 5 mM MgCI?. and 50 mM HEPES pH 7.5. and 1 .25 M D-arabinose. The reaction was carried out in triplicates. The formation of BTO was followed over time by withdrawing 20 pl of aliquot at certain time intervals. The sample was diluted 25-fold using 5 mM H2SO4 prior to filtration through 10 KDa spin column (VWR. Germany). The filtrate was analyzed by HPLC using an ion-exclusion column (RezexROA-Organic Acid H+(8%. Phenomenex. Germany) run isocratically using 2.5 mM H2SO4 at 70 °C for 20 min.

Table 3: Enzyme amounts for biotransformation of D-arabinose to (S)-BTO

*STY or space-time yield is defined as the volumetric productivity of (S)-BTO production. SP or specific productivity is defined as the amount of (S)-BTO formed per hour per g of total biocatalyst used.

Biotransformation of D-ribose to (S)-1,2,4-butanetriol The cell-free bioproduction of BTO was performed in 500 pl scale in Eppendorf tube. The solution contained combination of enzymes as shown in Table 4. TDP 0.1 mM. 5 mM MgCI?. and 50 mM HEPES pH 7.5. and 1.25 M D-ribose. The reaction was carried out in triplicates. The formation of BTO was followed over time by withdrawing 20 pl of aliquot at certain time intervals. The sample was diluted 25-fold using 5 mM H2SO4 prior to filtration through 10 KDa spin column (VWR. Germany). The filtrate was analyzed by HPLC using an ion-exclusion column (RezexROA-Organic Acid H+(8%. Phenomenex. Germany) run isocratically using 2.5 mM H2SO4 at 70 °C for 20 min.

Table 4: Enzyme amounts for biotransformation of D-ribose to (S)-BTO

*STY or space-time yield is defined as the volumetric productivity of (S)-BTO production. SP or specific productivity is defined as the amount of (S)-BTO formed per hour per g of total biocatalyst used.

Biotransformation of D-lyxose to (S)-1 ,2,4-butanetriol

The cell-free bioproduction of BTO was performed in 500 pl scale in Eppendorf tube. The solution contained combination of enzymes as shown in Table 5. TDP 0.1 mM. 5 mM MgCL and 50 mM HEPES pH 7.5. and 1.25 M D-lyxose. The reaction was carried out in triplicates. The formation of BTO was followed over time by withdrawing 20 pl of aliquot at certain time intervals. The sample was diluted 25-fold using 5 mM H2SO4 prior to filtration through 10 KDa spin column (VWR. Germany). The filtrate was analyzed by HPLC using an ion-exclusion column (RezexROA-Organic Acid H+(8%. Phenomenex. Germany) run isocratically using 2.5 mM H2SO4 at 70 °C for 20 min.

Table 5: Enzyme amounts for biotransformation of D-lyxose to (S)-BTO

*STY or space-time yield is defined as the volumetric productivity of (S)-BTO production. SP or specific productivity is defined as the amount of (S)-BTO formed per hour per g of total biocatalyst used.

Biotransformation of D-xylose to (S)-BTO with different lactonase concentrations

The progress of the biotransformation over time was monitored. Three different concentrations of A/mLacll (0, 2.5, 10 pM) (SEQ ID NO: 7) were applied. The initial concentration of D-xylose was 180 g/L (1.2 M), almost three-fold higher than the highest reported D-xylose used in literatures (Hu, S.; Gao, Q.; Wang, X.; Yang, J.; Xu, N.; Chen, K.; Xu, S.; Ouyang, P. Efficient Production of D-1 ,2,4-Butanetriol from D-Xylose by Engineered Escherichia Coli Whole-Cell Biocatalysts. 2018, 72 (4), 772-779). After following the biotransformation of D-xylose to (S)- BTO, it was observed that the addition of 2.5 pM was already sufficient, as the STY was similar to that of with 10 pM (Fig. 2). The average STY achieved from the both reactions is already 3.7 g/L/h, stands in contrast to 0.5 g/L/h, the highest reported STY reported by Hu et al. At the end of biotransformation a yield of >97% (mol/mol) was achieved from the reaction with addition of either 2.5 pM or 10 pM lactonase.

Table 6: Biotransformation of D-xylose to (S)-BTO with different lactonase concentrations

Boosting by intermediate to increase STY or lower cofactor consumption The relationship of co-factor-intermediate relationship to STY was tested. Among intermediates in the BTO process, D-xylonate can be relatively cheaply and efficiently produced. D-Xylonate is produced by oxidizing D-xylose, by means of either microorganisms, chemical catalyst, or electrochemistry. The cascade was run with different amounts of NAD ⁺ with and without addition of D-xylonate.

The results suggest that the amount of NAD ⁺ used greatly correlates to the STY (Fig. 3). For example, reducing NAD ⁺ from 3.3 g/L (5 mM) to 1.7 g/L (50% reduction) or to 0.7 (80% reduction) reduced up to 26% and 58% of the original STY (3.7 g/L/h), respectively. However, addition of D-xylonate can be used to compensate the decrease of STY. By adding 4.1 g/L (25 mM) D-xylonate, NAD ⁺ consumption can be reduced from 3.3 g/L to 0.33 g/L (>90% reduction) to still achieve the same STY (comparative line in Fig. 3). When the same amounts of NAD ⁺ are used, addition of 4.1 g/L D-xylonate boosted the average STY by 2 to 4-fold. The boosting effect of the intermediate is more pronounced at lower amounts of NAD ⁺ .

The highest STY was achieved when 3.3 g/L NAD ⁺ and 4.1 g/L D-xylonate were used in combination. In this condition, the average STY reached 6.8 g/L/h (Fig. 4B), 9.4 g/L/h in the first 9 h biotransformation (Fig. 4B) and conversion was the fastest. Without the intermediate addition the highest STY was achieved with the highest NAD ⁺ concentration 3.3 g/L (Fig. 4A) resulting in the fastest conversion.