WO2024100241A1 - Préparation biocatalytique de polyols issus de pentoses - Google Patents

Préparation biocatalytique de polyols issus de pentoses Download PDF

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WO2024100241A1
WO2024100241A1 PCT/EP2023/081405 EP2023081405W WO2024100241A1 WO 2024100241 A1 WO2024100241 A1 WO 2024100241A1 EP 2023081405 W EP2023081405 W EP 2023081405W WO 2024100241 A1 WO2024100241 A1 WO 2024100241A1
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pentose
butanetriol
lactonase
xylose
dehydrogenase
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PCT/EP2023/081405
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André Pick
Volker Sieber
Samuel SUTIONO
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Cascat Gmbh
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01175—D-Xylose 1-dehydrogenase (1.1.1.175)
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01081—Quorum-quenching N-acyl-homoserine lactonase (3.1.1.81)
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01009—Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase

Definitions

  • 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 is a versatile chemical with various applications.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention in a first aspect 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.
  • 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.
  • 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 is for convenience.
  • 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.
  • 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.
  • 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.
  • the pentose can be a mixture of different pentoses.
  • the pentose is an aldopentose.
  • 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.
  • 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.
  • the lactonase is present in an amount of from 0.01 to 20 pM and more preferably from 0.1 to 10 pM.
  • 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.
  • the co-factor applied in step a) is NAD + and/or NADH/H + .
  • the co-factor is recycled in the process.
  • 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.
  • the reaction solution can also run through the reactor containing the enzymes for increased conversion and yields several times.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 Caulo
  • 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.
  • 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.
  • the Space-Time-Yield 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.
  • STY Space-Time-Yield
  • 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.
  • the temperature in step b) is between 10 - 100 °C, more preferably between 20 - 90 °C and even more preferably between 20 - 80 °C.
  • the pH of the composition in the inventive process is between 3 to 12, and preferably 4 to 10.
  • Suitable buffers are known and include conventional buffers (systems), for example, acetate, potassium phosphate, Tris-HCI, glycylglycine and glycine buffers, or mixtures of these.
  • 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 .
  • the biocatalysts ions e.g. Mg 2+
  • the use of stabilizers, glycerol etc. may allow longer use of biocatalysts.
  • the pH of the composition is between 3 to 12 and more preferably 4 to 10.
  • the inventive process comprises, preferably consists of, the following steps in the following order
  • step (ii) a lactonase is used.
  • 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.
  • At least one of the intermediates, preferably the first intermediate is added to the process.
  • Adding an intermediate has the effect that the Space-Time Yield (STY) is increased while the amount of co-factor can be reduced.
  • STY Space-Time Yield
  • this effect of adding an intermediate is even more impressive.
  • 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).
  • cofactor price is one of the most contributing costs for in vitro enzymatic biotransformation this effect is advantageous.
  • STY Space-Time Yield
  • 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
  • the inventive process is cell-free.
  • the invention in a second aspect relates to a composition for converting a pentose into 1 ,2,4- butanetriol comprising, preferably consisting of,
  • 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
  • a third aspect of the invention relates to an aqueous composition
  • 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
  • a fourth aspect of the present invention relates to an aqueous composition according to the invention obtainable by the inventive process.
  • 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.
  • 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.
  • a lactonase preferably a lactonase derived from Noviherbaspirillum massiliense
  • composition for converting a pentose into 1 ,2,4-butanetriol and the aqueous composition also apply to this aspect where applicable.
  • 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.
  • 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.
  • D-Xylose, L-Arabinose, D-Arabinose, D-Ribose, D-Lyxose were obtained commercially.
  • 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..
  • 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.
  • 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.
  • 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.
  • (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé de conversion d'un pentose en 1,2,4-butanétriol comprenant les étapes suivantes : a) ajout à une composition comprenant de l'eau, d'au moins un cofacteur et d'un pentose, d'au moins cinq enzymes, et b) conversion enzymatique ultérieure du pentose en 1,2,4-butanétriol en présence d'au moins cinq enzymes, à l'étape a) au moins cinq enzymes étant choisies dans le groupe constitué de la déshydrogénase, de la déshydratase, de la lactonase, de la décarboxylase et des combinaisons de celles-ci, et au moins une enzyme de l'étape a) étant une lactonase.
PCT/EP2023/081405 2022-11-11 2023-11-10 Préparation biocatalytique de polyols issus de pentoses WO2024100241A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US7923226B2 (en) 2003-10-01 2011-04-12 Board Of Trustees Of Michigan State University Synthesis of 1,2,4-butanetriol enantiomers from carbohydrates
US20110165641A1 (en) * 2006-03-31 2011-07-07 The Board Of Trustees Of Michigan State University Synthesis of 1,2,4-Butanetriol Enantiomers from Carbohydrates

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US7923226B2 (en) 2003-10-01 2011-04-12 Board Of Trustees Of Michigan State University Synthesis of 1,2,4-butanetriol enantiomers from carbohydrates
US20110165641A1 (en) * 2006-03-31 2011-07-07 The Board Of Trustees Of Michigan State University Synthesis of 1,2,4-Butanetriol Enantiomers from Carbohydrates

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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, vol. 12, no. 4, 2018, pages 772 - 779
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