Biosynthesis method of C6-C8 medium-chain 1,2-diol and its recombinant microorganism

Abstract

The present invention belongs to the field of metabolic engineering, and relates to a biosynthetic method of a medium-chain 1,2-diol of C6-C8 and a recombinant microorganism thereof. The present invention provides a non-naturally occurring microorganism, which has a medium-chain 1,2-diol synthesis pathway with a chain length of C6-C8, so that a medium-chain 1,2-diol with a chain length of C6-C8 can be synthesized in vivo. The metabolic pathway includes the following enzymes: carboxylic acid reductase, ketoreductase, acyl CoA transferase or acyl CoA ligase, cinnamic acid acylase, acyl CoA reductase, aldehyde reductase, etc. The recombinant microorganism of the present invention is fermented and cultured with organic carbon sources such as glucose in a shake flask, and 243.2 mg / L 1,2-hexanediol, 83.6 mg / L 1,2-heptanediol and 49.7 mg / L 1,2-octanediol can be obtained. The method is simple to operate, green and environmentally friendly, suitable for large-scale industrial synthesis, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering and relates to a biosynthesis method for a C6-C8 medium-chain 1,2-diol and a recombinant microorganism thereof. Background Art

[0002] 1,2-Diols are an important class of bulk chemicals, which are widely used in polymer materials, cosmetics, fuels, food and pharmaceutical industries, especially in solvents, moisturizers, preservatives and other aspects. The existing method of synthesizing diols by microorganisms uses renewable raw materials, reduces dependence on fossil resources and reduces carbon dioxide emissions, which is of great significance. This method is easy to operate and suitable for large-scale production. It has received widespread attention at home and abroad in recent years. However, this method still faces challenges. On the one hand, the current microbial synthesis method mainly focuses on the synthesis of short-chain C2-C5 diols; on the other hand, there is a lack of effective natural synthesis pathways and low yields of genetically engineered bacteria.

[0003] Medium-chain (C6-C8) 1,2-diols, including 1,2-hexanediol, 1,2-heptanediol and 1,2-octanediol, play an irreplaceable role in high-performance modified plastics in addition to their applications in moisturizers, preservatives, etc., and are widely used in industry. It is crucial to find a method suitable for industrial large-scale synthesis of medium-chain (C6-C8) 1,2-diols.

[0004] At present, the production methods of 1,2-hexanediol include chemical method and biological synthesis method. The chemical method is divided into ethylene direct oxidation method and oxirane hydrolysis method according to different reactants, but this method relies on fossil raw materials and pollutes the environment. The patent with publication number CN117487862A discloses a biological method for synthesizing 1,2-hexanediol, which uses n-hexanoic acid as the starting material and generates the final product under the action of P450 monooxygenase and lipase. It has the advantages of mild conditions, green environmental protection and low cost, but this method requires additional addition of enzymes, H2O2, butanol and other substances, and the operation is complicated, which is not conducive to industrial production.

[0005] In summary, the prior art lacks a simple, environmentally friendly, and industrially large-scale synthesis method for producing medium-chain (C6-C8) 1,2-diols. Therefore, it is urgent to develop a biosynthetic pathway and recombinant microorganism for producing medium-chain (C6-C8) 1,2-diols. Summary of the invention

[0006] In order to overcome the defects of the prior art, the present invention provides a method for producing 1,2-hexanediol based on microbial fermentation, and applies the method to the biosynthesis of 1,2-heptanediol and 1,2-octanediol.

[0007] The present invention provides a biosynthetic method for a medium-chain 1,2-diol with a chain length of C6-C8, which comprises the following steps:

[0008] A) Reduction of 2-oxocarboxylic acids to 2-hydroxycarboxylic acids;

[0009] B) Reduction of 2-hydroxycarboxylic acids to 2-hydroxyaldehydes;

[0010] C) Reduction of 2-hydroxyaldehydes to 1,2-diols;

[0011] The chain length of the 2-oxocarboxylic acid is C6-C8.

[0012] Further, step A) is to convert 2-oxocarboxylic acid into 2-hydroxycarboxylic acid using 2-oxocarboxylic acid reductase;

[0013] And / or, the method adopted in step B) is any one of the following methods:

[0014] Method 1: 2-Hydroxycarboxylic acid is reduced to 2-hydroxyaldehyde by carboxylic acid reductase;

[0015] or,

[0016] Method 2:

[0017] B1) 2-hydroxycarboxylic acid is converted to 2-hydroxyacyl-CoA by acyl CoA transferase or acyl CoA ligase;

[0018] B2) 2-hydroxyacyl-CoA is reduced to 2-hydroxyaldehyde by acyl-CoA reductase; preferably, cinnamic acylase is also added to B1);

[0019] And / or, step C) is to reduce the 2-hydroxyaldehyde to 1,2-diol using aldehyde reductase.

[0020] Furthermore, carboxylate reductase requires an activating factor.

[0021] Furthermore, the raw material 2-oxocarboxylic acid used in the method of the present invention can be prepared according to the following method:

[0022] Step 1.1, threonine is deaminated and dehydrated to generate 2-oxobutyric acid, or pyruvate is condensed with one molecule of acetyl-CoA to generate 2-oxobutyric acid;

[0023] Step 1.2, 2-oxobutyric acid is condensed with acetyl coenzyme A, dehydrated, hydroxylated, oxidized, and decarboxylated, and the reaction is repeated again to obtain the product.

[0024] Preferably, threonine is converted into 2-oxobutyrate using threonine dehydratase;

[0025] and / or, using citramalate synthase to condense pyruvate with one molecule of acetyl-CoA to generate 2-oxobutyrate;

[0026] And / or, a combination of isopropylmaleate synthase, 3-isopropylmalate dehydrogenase and 3-isopropylmalate dehydratase is used to convert 2-oxobutyrate into 2-oxocarboxylic acid.

[0027] Furthermore, the biosynthesis method is reduction and conversion under at least one of the conditions of whole cells, immobilized enzymes, a combination of purified enzymes, and crude enzyme solution.

[0028] Furthermore, the whole cell is a recombinant microorganism; the starting strain of the recombinant microorganism is selected from Bacillus subtilis, Corynebacterium glutamicum, Yarrowia lipolytica, Saccharomyces cerevisiae or Escherichia coli.

[0029] Further, the 2-oxocarboxylate reductase is encoded by one or more genes selected from the group consisting of ldh, ldhA, panE, ara, ara1, ara2, araDH, YGL039W, adh, CXQ87_003259;

[0030] and / or, the carboxylate reductase is encoded by the Car gene, and the activator is encoded by the sfp gene;

[0031] and / or, the acyl-CoA transferase or acyl-CoA ligase is encoded by one or more genes selected from the group consisting of fldA, hadA, pct, Cat2, carB, 4CL, PaaK, Cat1, PGN_0725, PGN_1341, PGN_1888, fadD;

[0032] and / or, the acyl-CoA reductase is encoded by one or more genes selected from the group consisting of maqu2220, maqu2507, bld, ald, pdcD, pduP, adhE, FAR, acr, hom, fadB, fadJ, hbd, phaB, phbB, mdh, ldh;

[0033] and / or, the cinnamate acylase is encoded by one or more genes selected from the group consisting of: Pt4CL, At4CL2, At4CL5, Os4CL, Pc4CL;

[0034] And / or, the aldehyde reductase is encoded by one or more genes selected from the group consisting of yqhD, ADH2, adh, bdh, yahK, adhA, 4-hbd, dhaT, mmsb, P84067, adhI, alrA, sucD, and bld.

[0035] Further, in the recombinant microorganism, threonine dehydratase is encoded by the ilvA gene; and / or, citramalate synthase is encoded by the cimA gene; and / or, a combination of isopropylmaleate synthase, 3-isopropylmalate dehydrogenase, and 3-isopropylmalate dehydratase is encoded by the leuABCD gene.

[0036] Furthermore, it also includes the above biosynthesis method by further increasing the intracellular precursor concentration or adding precursors in vitro such as threonine, pyruvate, cofactors NAD(P)H, NAD(P) + , ATP, etc. to further increase the production of medium-chain 1,2-diols with a chain length of C6-C8.

[0037] Further, recombinant microorganisms expressing ldh, yqhD, fldA, maqu2507, and Pc4CL genes are cultured in a medium including cinnamic acid, expression is induced, 2-oxocarboxylic acid is added, and fermentation is continued to obtain 1,2-diol;

[0038] Alternatively, a recombinant microorganism expressing the ilvA, ldh, zwf, yqhD, fldA, maqu2507, Pc4CL, and leuABCD genes is cultured in a medium containing cinnamic acid, expression is induced, threonine is added, and fermentation is continued to obtain 1,2-diol;

[0039] Alternatively, a recombinant microorganism expressing cimA, ldh, zwf, yqhD, fldA, maqu2507, Pc4CL, and leuABCD genes is cultured in a medium comprising glucose and cinnamic acid, expression is induced, and fermentation is continued to obtain 1,2-diol.

[0040] In the fermentation method of the present invention, the culture conditions are: 37°C, 200rpm rotation speed, adding an inducer to the culture medium, fermenting for 48 hours, and obtaining the target product. The pH of the culture medium used for the culture is 6.8, and the formula of the culture medium includes 20g / L glucose, 12.8g / L Na2HPO4·7H2O, 3g / L KH2PO4, 0.5g / L NaCl, 1g / L NH4Cl, 0.5g / LMgSO4, 0.5g / L cinnamic acid, 5g / L yeast powder, 0.01g / L CaCl2, 100mg / L ampicillin, and 35mg / L chloramphenicol; the inducer used for fermentation is selected from isopropyl-β-D-thiogalactoside.

[0041] The present invention also provides a product obtained by any one of the above biosynthetic methods.

[0042] The present invention provides a recombinant microorganism, wherein the recombinant microorganism overexpresses genes for 2-oxocarboxylic acid reductase, carboxylic acid reductase, acyl CoA reductase, acyl CoA transferase, acyl CoA ligase, cinnamate acylase and / or aldehyde reductase;

[0043] Preferably, the starting strain is selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae or Yarrowia lipolytica.

[0044] Furthermore, the recombinant microorganism overexpresses genes for 2-oxocarboxylic acid reductase, carboxylic acid reductase, and aldehyde reductase;

[0045] Or, overexpressing genes of 2-oxocarboxylic acid reductase, acyl-CoA reductase, cinnamate acylase, acyl-CoA transferase or acyl-CoA ligase, aldehyde reductase.

[0046] Further, the 2-oxocarboxylate reductase is encoded by one or more genes selected from the group consisting of ldh, ldhA, panE, ara, ara1, ara2, araDH, YGL039W, adh, CXQ87_003259;

[0047] and / or, the carboxylate reductase is encoded by a CAR gene;

[0048] and / or, the acyl-CoA reductase is encoded by one or more genes selected from the group consisting of maqu2220, maqu2507, bld, ald, pdcD, pduP, adhE, FAR, acr, hom, fadB, fadJ, hbd, phaB, phbB, mdh, ldh;

[0049] and / or, the acyl-CoA transferase or acyl-CoA ligase is encoded by one or more genes selected from the group consisting of fldA, hadA, pct, Cat2, carB, 4CL, PaaK, Cat1, PGN_0725, PGN_1341, PGN_1888, fadD;

[0050] and / or, the cinnamate acylase is encoded by one or more genes selected from the group consisting of: Pt4CL, At4CL2, At4CL5, Os4CL, Pc4CL;

[0051] And / or, the aldehyde reductase is encoded by one or more genes selected from the group consisting of yqhD, ADH2, adh, bdh, yahK, adhA, 4-hbd, dhaT, mmsb, P84067, adhI, alrA, sucD, and bld.

[0052] The present invention provides a non-naturally occurring recombinant microorganism having a medium-chain 1,2-diol synthesis pathway with a chain length of C6-C8 ( Figure 1 ), so that medium-chain 1,2-diols with a chain length of C6-C8 can be synthesized in vivo (hereinafter referred to as medium-chain 1,2-diols). The metabolic pathway includes the following enzymes: amino acid dehydratase, citramalate synthase, isopropylmaleate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate dehydratase, carboxylic acid reductase, ketoreductase, acyl CoA transferase, cinnamate acylase, acyl CoA ligase, acyl CoA reductase, aldehyde reductase, etc.

[0053] The recombinant microorganism of the present invention can be fermented and cultured with an organic carbon source such as glucose in a shake flask to obtain 243.2 mg / L 1,2-hexanediol, 83.6 mg / L 1,2-heptanediol and 49.7 mg / L 1,2-octanediol. A biological method for synthesizing medium-chain 1,2-diols is provided. The method is simple to operate, green and environmentally friendly, suitable for industrial large-scale synthesis, and has good application prospects.

[0054] In some embodiments, the invention is directed to a method for producing a 1,2-medium chain diol, the method comprising culturing such a non-naturally occurring recombinant microorganism under conditions and for a sufficient period of time to produce the 1,2-medium chain diol.

[0055] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0056] "2-Oxocarboxylic acid" refers to a saturated carbon chain containing 6-8 carbon atoms and comprising a carboxylic acid and a carbonyl group, wherein the carbon atom of the carboxylic acid is at one end of the carbon chain and the carbon atom of the carbonyl group is adjacent to the carbon atom of the carboxylic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Microbial synthesis pathways for medium-chain 1,2-diols with chain lengths of C6-C8

[0058] Figure 2 Plasmid pSTV28-ldh-zwf

[0059] Figure 3 Plasmid pTrc99a-yqhD-sfp-car

[0060] Figure 4 Plasmid pTrc99a-yqhD-maqu2507-fldA

[0061] Figure 5 Plasmid pTrc99a-yqhD-maqu2507

[0062] Figure 6 Plasmid pTrc99a-yqhD-maqu2507-Ptrc-fldA-Pc4CL

[0063] Figure 7 Plasmid pSTV28-leuABCD

[0064] Figure 8 Plasmid pSTV28-leuABCD-Ptrc-ilvA-ldh-zwf

[0065] Fig. 9 Plasmid pSTV28-leuABCD-Ptrc-cimA-ldh-zwf DETAILED DESCRIPTION

[0066] The present invention is directed in part to a non-naturally occurring recombinant microorganism that expresses genes encoding enzymes that catalyze the production of 1,2-medium chain (C6-C8) diols. The present invention discloses two pathways for producing 1,2-medium chain diols, such as Figure 1 As shown: 1) After 2-oxocarboxylic acid is reduced to 2-hydroxycarboxylic acid, it is directly reduced to 2-hydroxyaldehyde and 1,2-diol; 2) After 2-oxocarboxylic acid is reduced to 2-hydroxycarboxylic acid, 2-hydroxyacyl-CoA is first formed and then reduced to 2-hydroxyaldehyde and 1,2-diol. At the same time, the synthesis of the intermediate product 2-oxocarboxylic acid in the body is based on two precursors: threonine and pyruvate.

[0067] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and replacements to the present invention without departing from the purpose and spirit of the present invention. In the following examples, the reagents and materials not particularly described are all commercially available products.

[0068] In the early stage, we screened acyl CoA reductase, acyl CoA transferase, and cinnamoyl CoA respectively. The screening methods are as follows:

[0069] Screening of acyl-CoA reductase:

[0070] The gene encoding the CoA transferase pct (SEQ ID NO.1, amino acid sequence WP_014015705.1), the gene encoding the aldehyde reductase yqhD (KEGG: JW2978), and five acyl-CoA reductases (ACRs) - maqu2507 (SEQ ID NO.2, amino acid sequence WP_011785966.1), maqu2220 (SEQ ID NO.3, amino acid sequence WP_011785687.1), bld (SEQ ID NO.4, amino acid sequence WP_07736660 5.1), ald (SEQ ID NO.5, amino acid sequence WP_077844109.1), pduP (SEQ ID NO.6, amino acid sequence WP_048265961.1)-one of the genes, respectively cloned into the pTrc99a vector under the control of the Ptrc promoter, and obtained five pTrc99a-yqhD-ACR-pct plasmids, which were transformed into Escherichia coli strain W3110 by electrotransformation with the plasmid pSTV28-ldh-zwf prepared in Example 1. The obtained strain was cultured in a fermentation medium at a fermentation temperature of 37 ° C and a rotation speed of 200 rpm. When the bacterial cell OD600 reached 0.8, 0.1 mM IPTG was added for induction, and 1 g / L 2-oxocarboxylic acid raw material was added at the same time. After continuing the fermentation for 48 hours, the diol product was detected by HPLC. When the acyl CoA reductase was maqu2507, the yield of the product was the highest.

[0071] The nucleotide sequences of pct, maqu2507, maqu2220, bld, ald, and pduP are shown in the following table:

[0072] Table 1

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Screening of acyl-CoA transferase:

[0079] The gene encoding CoA reductase maqu2507 (SEQ ID NO.2), the gene encoding aldehyde reductase yqhD (KEGG: JW2978), and five acyl-CoA transferases (CTs) - fldA (SEQ ID NO.7, amino acid sequence WP_108429007.1), hadA (SEQ ID NO.8, amino acid sequence WP_095908939.1), pct (SEQ ID NO.1), Cat2 (SEQ ID NO.9, amino acid sequence WP_005873621.1), carB (SEQ ID NO.10, amino acid sequence WP_014355956.1)-one of the genes, respectively cloned into the pTrc99a vector under the control of the Ptrc promoter, and obtained five pTrc99a-yqhD-maqu2507-CT plasmids, which were respectively transformed into the Escherichia coli strain W3110 with the plasmid pSTV28-ldh-zwf prepared in Example 1 by chemical transformation. The obtained strain was cultured in a fermentation medium, the fermentation temperature was 37 ° C, the speed was 200 rpm, and when the bacterial cell OD600 reached 0.8, 0.1 mM IPTG was added for induction, and 1 g / L 2-oxocarboxylic acid raw material was added at the same time. After continuing the fermentation for 48 hours, the diol product was detected by HPLC. When the acyl CoA transferase is fldA (SEQ ID NO.7), the yield of the product is the highest.

[0080] The nucleotide sequences of fldA, hadA, Cat2, and carB are shown in the following table:

[0081] Table 2

[0082]

[0083]

[0084]

[0085]

[0086] Screening of Cinnamoyl CoA:

[0087] The yqhD-maqu2507 fragment was PCR amplified from pTrc99a-yqhD-maqu2507-fldA prepared in Example 2 and cloned into the pTrc99a vector under the control of the Ptrc promoter to obtain pTrc99a-yqhD-maqu2507. The genes of five cinnamic acylases 4CL, namely Pt4CL (SEQ ID NO.11, amino acid sequence AAA92669.1), At4CL2 (SEQ ID NO.12, amino acid sequence NP_188761.1), At4CL5 (SEQ ID NO.13, amino acid sequence NP_188760), Os4CL (SEQ ID NO.14, amino acid sequence NP_001396278.1), and Pc4CL (SEQ ID NO.15, amino acid sequence P14912), were cloned into a Ptrc promoter by overlapping extension PCR with fldA, and then cloned into the linearized vector pTrc99a-yqhD-maqu2507 to obtain five pTrc99a-yqhD-maqu2507-Ptrc-fldA-4CL plasmids. This group of plasmids were co-transformed into Escherichia coli strain W3110 with the plasmid pSTV28-ldh-zwf prepared in Example 1 by electrotransformation. The obtained strain was cultured in a fermentation medium containing cinnamic acid, the fermentation temperature was 37°C, the rotation speed was 200rpm, and when the bacterial cell OD600 reached 0.8, 0.1mM IPTG was added for induction, and 1g / L 2-oxocarboxylic acid raw material was added at the same time. After continuing fermentation for 48 hours, the diol product was detected by HPLC. When the cinnamic acid acylase was Pc4CL (SEQ ID NO.15), the yield of the product was the highest.

[0088] Therefore, maqu2507 is the preferred acyl-CoA reductase, fldA is the preferred acyl-CoA transferase, and Pc4CL is the preferred cinnamate acylase.

[0089] The nucleotide sequences of Pt4CL, At4CL2, At4CL5, Os4CL and Pc4CL are shown in the following table:

[0090] Table 3

[0091]

[0092]

[0093]

[0094]

[0095] Example 1 Direct reduction of 2-oxocarboxylic acids (C6-C8) to form 1,2-diols

[0096] The conversion of 2-oxocarboxylic acids to 1,2-diols can be achieved via a three-step reaction: 2-oxocarboxylic acids are converted to 2-hydroxycarboxylic acids by ketoreductase, which are then reduced to 2-hydroxyaldehydes by carboxylic acid reductase, and then reduced to 1,2-diols by 2-hydroxyaldehyde reductase.

[0097] In this example, Escherichia coli was used as a host microorganism for strain transformation, and the nucleic acid encoding the above enzyme was expressed in Escherichia coli using existing molecular biological techniques. Specifically, lactate dehydrogenase ldh of Lactobacillus casei (Table 4, SEQ ID NO.16, amino acid sequence BAN75276.1) and glucose-6-phosphate 1-dehydrogenase zwf of Escherichia coli were cloned into the pSTV28 vector to obtain the pSTV28-ldh-zwf plasmid (such as Figure 2 At the same time, the gene encoding carboxylic acid reductase car (Table 5, SEQ ID NO.17, amino acid sequence WP_005082584.1) and its activating factor gene sfp (EU882341.1), and the gene encoding aldehyde reductase yqhD (KEGG: JW2978) were cloned into the pTrc99a vector under the control of the Ptrc promoter to obtain the plasmid pTrc99a-yqhD-sfp-car (such as Figure 3 These two plasmids were then transformed into E. coli strain W3110 by chemical transformation to overexpress the relevant enzymes from 2-oxocarboxylic acids to 1,2-diols.

[0098] Table 4

[0099]

[0100]

[0101]

[0102] The obtained strain was cultured in a fermentation medium at a fermentation temperature of 37°C and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction, and 1 g / L 2-oxohexanoic acid, 1 g / L 2-oxoheptanoic acid or 1 g / L 2-oxooctanoic acid precursor was added respectively. After continuing the fermentation for 48 hours, HPLC was used to detect the products of 1,2-hexanediol, 1,2-heptanediol and 1,2-octanediol, and the yields of 97.5 mg / L 1,2-hexanediol, 34.6 mg / L 1,2-heptanediol and 17.3 mg / L 1,2-octanediol were obtained, respectively.

[0103] The formula of fermentation medium is (g / L): glucose 20, Na2HPO4·7H2O 12.8, KH2PO4 3, NaCl 0.5, NH4Cl1, MgSO4 0.5, yeast powder 5, CaCl2 0.01, antibiotics (mg / L): ampicillin 100, chloramphenicol 35.

[0104] Example 2 Reduction of 2-oxohexanoic acid to 1,2-hexanediol via acyl-CoA

[0105] In this example, the conversion of 2-oxohexanoic acid to 1,2-hexanediol is achieved through a four-step reaction: 2-oxohexanoic acid is converted to 2-hydroxyhexanoic acid by ketoreductase, the latter is catalyzed by CoA transferase or CoA ligase to form 2-hydroxyhexanoyl CoA, then reduced by acyl CoA reductase to form 2-hydroxyhexanal, and finally reduced by 2-hydroxyhexanal reductase to form 1,2-hexanediol.

[0106] In this example, E. coli was used as a host microorganism for strain transformation, and the nucleic acid encoding the above enzyme was expressed in E. coli using existing molecular biological techniques. In this example, the preferred acyl CoA reductase and CoA transferase combination maqu2507 and fldA were used for testing.

[0107] The gene encoding CoA transferase fldA (SEQ ID NO.7), the gene encoding aldehyde reductase yqhD (KEGG: JW2978), and the gene maqu2507 (SEQ ID NO.2) encoding acyl CoA reductase (ACR) were cloned into the pTrc99a vector under the control of the Ptrc promoter to obtain the pTrc99a-yqhD-maqu2507-fldA plasmid (as Figure 4 ), and co-transformed into Escherichia coli strain W3110 by electrotransformation and the pSTV28-ldh-zwf plasmid prepared in Example 1 to overexpress the relevant enzymes from 2-oxohexanoic acid to 1,2-hexanediol.

[0108] The obtained strain was cultured in a fermentation medium at a fermentation temperature of 37°C and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction, and 1 g / L 2-oxohexanoic acid precursor was added at the same time. After continuing the fermentation for 48 hours, the 1,2-hexanediol product was detected by HPLC, and a yield of 33.4 mg / L was obtained.

[0109] The formula of fermentation medium is (g / L): glucose 20, Na2HPO4·7H2O 12.8, KH2PO4 3, NaCl 0.5, NH4Cl 1, MgSO4 0.5, yeast powder 5, CaCl2 0.01, antibiotics (mg / L): ampicillin 100, chloramphenicol 35.

[0110] Example 3 Increasing 1,2-diol production by overexpressing cinnamic acylase

[0111] In this example, the conversion of 2-oxocarboxylic acid to 1,2-diol is achieved through a four-step reaction: 2-oxocarboxylic acid is converted into 2-hydroxycarboxylic acid by ketoreductase, the latter is catalyzed by CoA transferase or CoA ligase to form 2-hydroxycarboxylic acid CoA, then reduced by acyl CoA reductase to form 2-hydroxyaldehyde, and finally reduced by 2-hydroxyaldehyde reductase to form 1,2-diol.

[0112] In this example, E. coli was used as a host microorganism for strain transformation, and the nucleic acid encoding the above enzyme was expressed in E. coli using existing molecular biological techniques. In this example, the preferred acyl CoA reductase, acyl CoA transferase-cinnamic acylase combination maqu2507 and fldA-Pc4CL combination were used for biosynthesis.

[0113] The gene encoding aldehyde reductase yqhD (KEGG: JW2978) and the gene maqu2507 (SEQ ID NO. 2) encoding acyl CoA reductase (ACR) were cloned into the pTrc99a vector under the control of the Ptrc promoter to obtain the pTrc99a-yqhD-maqu2507 plasmid (as Figure 5 ). The gene of CoA transferase fldA (SEQ ID NO.7) and cinnamic acylase Pc4CL gene (SEQ ID NO.15) were cloned into a Ptrc promoter by overlap extension PCR, and then cloned into the linearized vector pTrc99a-yqhD-maqu2507 to obtain plasmid pTrc99a-yqhD-maqu2507-Ptrc-fldA-Pc4CL (such as Figure 6 ). This plasmid and the pSTV28-ldh-zwf plasmid prepared in Example 1 were co-transformed into Escherichia coli strain W3110 by electrotransformation to overexpress the enzymes related to the conversion of 2-oxocarboxylic acid to 1,2-diol.

[0114] The obtained strain was cultured in a fermentation medium at a fermentation temperature of 37°C and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction, and 1 g / L 2-oxohexanoic acid, 1 g / L 2-oxoheptanoic acid or 1 g / L 2-oxooctanoic acid precursor was added respectively. After continuing the fermentation for 48 hours, 1,2-hexanediol, 1,2-heptanediol and 1,2-octanediol products were detected by HPLC, and 243.2 mg / L 1,2-hexanediol, 86.3 mg / L 1,2-heptanediol and 49.7 mg / L 1,2-octanediol yields were obtained, respectively.

[0115] The fermentation medium formula is (g / L): glucose 20, Na2HPO4·7H2O 12.8, KH2PO4 3, NaCl 0.5, NH4Cl 1, MgSO4 0.5, cinnamic acid 0.5, yeast powder 5, CaCl2 0.01, pH 6.8. Antibiotics (mg / L): ampicillin 100, chloramphenicol 35.

[0116] Example 4 Synthesis of C6-C8 1,2-diols from threonine

[0117] In this example, the conversion of threonine to the precursor 2-oxocarboxylic acid can be achieved through multiple pathways in five enzymatic steps (e.g. Figure 1 Threonine is deaminated and dehydrated to form 2-oxobutyric acid, which is then condensed with a molecule of acetyl-CoA, dehydrated, hydroxylated, oxidized, and decarboxylated to form 2-oxopentanoic acid; 2-oxopentanoic acid can enter the same cycle multiple times to form 2-oxohexanoic acid, 2-oxoheptanoic acid, and 2-oxooctanoic acid.

[0118] In this example, E. coli was used as a host microorganism for strain transformation, and the nucleic acid encoding the above enzyme was expressed in E. coli using existing molecular biological techniques. Specifically, the E. coli gene cluster leuABCD (Table 5, SEQ ID NO. 18) was cloned into the pSTV28 vector to obtain the pSTV28-leuABCD plasmid (eg Figure 7 At the same time, the coding genes of Escherichia coli threonine dehydratase ilvA (KEGG: JW3745), Lactobacillus casei lactate dehydrogenase ldh (SEQ ID NO.16) and Escherichia coli glucose-6-phosphate 1-dehydrogenase zwf were cloned into a Ptrc promoter by overlap extension PCR, and then the fragment was inserted into the linearized plasmid pSTV28-leuABCD by one-step cloning to obtain plasmid pSTV28-leuABCD-Ptrc-ilvA-ldh-zwf (such as Figure 8 ), the plasmid and the pTrc99a-yqhD-sfp-car plasmid prepared in Example 1 were transformed into Escherichia coli strain W3110 by electroporation to overexpress the enzymes related to threonine to 1,2-diol.

[0119] Table 5

[0120]

[0121]

[0122]

[0123] The obtained strain was cultured in a fermentation medium (same as the medium formula in Example 1) at a fermentation temperature of 37° C. and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction, and 3 g / L threonine precursor was added at the same time. After continuing the fermentation for 48 hours, the 1,2-diol product was detected by HPLC, and 40.3 mg / L 1,2-hexanediol, 10.4 mg / L 1,2-heptanediol and 3.1 mg / L 1,2-octanediol yields were obtained.

[0124] Example 5 Synthesis of C6-C8 1,2-diols from Threonine via Acyl-CoA Reduction

[0125] In this example, the conversion of threonine to the precursor 2-oxocarboxylic acid can be achieved through multiple pathways in five enzymatic steps (e.g. Figure 1 Threonine is deaminated and dehydrated to form 2-oxobutyric acid, which is then condensed with a molecule of acetyl-CoA, dehydrated, hydroxylated, oxidized, and decarboxylated to form 2-oxopentanoic acid; 2-oxopentanoic acid can enter the same cycle multiple times to form 2-oxohexanoic acid, 2-oxoheptanoic acid, and 2-oxooctanoic acid.

[0126] In this example, Escherichia coli was used as a host microorganism for strain modification, and the nucleic acid encoding the above enzyme was expressed in Escherichia coli using existing molecular biological techniques. Specifically, the plasmid pSTV28-leuABCD-Ptrc-ilvA-ldh-zwf obtained in Example 4 and the plasmid pTrc99a-yqhD-maqu2507-Ptrc-fldA-Pc4CL obtained in Example 3 were transformed into Escherichia coli strain W3110 by electroporation to overexpress the enzymes related to threonine to 1,2-diol.

[0127] The obtained strain was cultured in a fermentation medium (same as the medium formula in Example 3) at a fermentation temperature of 37°C and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction, and 3 g / L threonine precursor was added at the same time. After continuing the fermentation for 48 hours, the 1,2-diol product was detected by HPLC, and 75.4 mg / L 1,2-hexanediol and 33.6 mg / L 1,2-heptanediol were obtained.

[0128] Example 6 Synthesis of C6-C8 1,2-diols from pyruvic acid

[0129] In this example, the conversion of pyruvate to the precursor 2-oxocarboxylic acid can be carried out via multiple pathways with five enzyme-catalyzed steps (e.g. Figure 1Pyruvate is condensed with one molecule of acetyl-CoA to form 2-oxobutyric acid, which is then condensed with another molecule of acetyl-CoA, dehydrated, hydroxylated, oxidized, and decarboxylated to form 2-oxopentanoic acid; 2-oxopentanoic acid can enter the same cycle multiple times to form 2-oxohexanoic acid, 2-oxoheptanoic acid, and 2-oxooctanoic acid.

[0130] In this example, Escherichia coli was used as a host microorganism for strain transformation, and the nucleic acid encoding the above enzymes was expressed in Escherichia coli using existing molecular biological techniques. Specifically, the coding genes of 2-methylmalate synthase cimA of Methanococcus jannaschii (Table 6, SEQ ID NO.19, amino acid sequence WP_010870909.1), lactate dehydrogenase ldh of Lactobacillus casei (SEQ ID NO.16) and glucose-6-phosphate 1-dehydrogenase zwf of Escherichia coli were cloned into a Ptrc promoter by overlapping extension PCR, and then the fragment was inserted into the linearized plasmid pSTV28-leuABCD by one-step cloning to obtain plasmid pSTV28-leuABCD-Ptrc-cimA-ldh-zwf (as shown in Figure 6). Fig. 9 ), the plasmid and the plasmid pTrc99a-yqhD-sfp-car prepared in Example 1 were transformed into Escherichia coli strain W3110 by electroporation to overexpress the enzymes related to the conversion of pyruvate to 1,2-diol.

[0131] Table 6

[0132]

[0133] The obtained strain was cultured in a fermentation medium (same as the medium formula in Example 1) at a fermentation temperature of 37° C. and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction. After continuing the fermentation for 48 hours, the 1,2-diol product was detected by HPLC, and 12.5 mg / L 1,2-hexanediol was obtained, while 1,2-heptanediol and 1,2-octanediol were not detected.

[0134] Example 7 Synthesis of C6-C8 1,2-diols from pyruvate via acyl-CoA reduction

[0135] In this example, the conversion of pyruvate to the precursor 2-oxocarboxylic acid can be carried out via multiple pathways with five enzyme-catalyzed steps (e.g. Figure 1 Pyruvate is condensed with one molecule of acetyl-CoA to form 2-oxobutyric acid, which is then condensed with another molecule of acetyl-CoA, dehydrated, hydroxylated, oxidized, and decarboxylated to form 2-oxopentanoic acid; 2-oxopentanoic acid can enter the same cycle multiple times to form 2-oxohexanoic acid, 2-oxoheptanoic acid, and 2-oxooctanoic acid.

[0136] In this example, Escherichia coli was used as a host microorganism for strain modification, and the nucleic acid encoding the above enzyme was expressed in Escherichia coli using existing molecular biology techniques. Specifically, the plasmid pSTV28-leuABCD-Ptrc-cimA-ldh-zwf prepared in Example 6 and the plasmid pTrc99a-yqhD-maqu2507-Ptrc-fldA-Pc4CL prepared in Example 3 were transformed into Escherichia coli strain W3110 by electroporation to overexpress the enzymes related to pyruvate to 1,2-diol.

[0137] The obtained strain was cultured in a fermentation medium (same as the medium formula in Example 3) at a fermentation temperature of 37° C. and a rotation speed of 200 rpm. When the OD600 of the bacteria reached 0.8, 0.1 mM IPTG was added for induction. After continuing the fermentation for 48 hours, the 1,2-diol product was detected by HPLC, and 27.4 mg / L 1,2-hexanediol was obtained, while 1,2-heptanediol and 1,2-octanediol were not detected.

[0138] The results of the above examples are shown in Table 7: 2-oxocarboxylic acids can be catalyzed to synthesize 1,2-diols through the ldh, zwf, sfp, car, and yqhD pathways; when sfp and car in the pathway are replaced by maqu2507 and fldA, 1,2-diols can also be catalyzed to synthesize. When Pc4CL is further introduced, the catalytic yields of maqu2507 and fldA can be significantly increased. When glucose is used as an organic carbon source and threonine is added, the recombinant microorganism can simultaneously catalyze the synthesis of 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol; when no raw materials are added and only organic carbon sources such as glucose are used as substrates, the recombinant microorganism can catalyze the synthesis of 1,2-hexanediol. When the fldA, maqu2507, and Pc4CL pathways are used, the yield of the product is significantly increased.

[0139] Table 7 Relationship between reaction pathway, product and yield

[0140]

[0141]

[0142] It can be seen from the above examples that the present invention provides a non-naturally occurring recombinant microorganism having a medium-chain 1,2-diol synthesis pathway with a chain length of C6-C8, thereby being able to synthesize medium-chain 1,2-diols in vivo. The metabolic pathway includes the following enzymes: amino acid dehydratase, citramalate synthase, isopropylmaleate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate dehydratase, carboxylic acid reductase, ketoreductase, acyl CoA transferase, cinnamic acid acylase, CoA ligase, acyl CoA reductase, aldehyde reductase, etc. The recombinant microorganism of the present invention can be fermented and cultured with organic carbon sources such as glucose in a shake flask to obtain 243.2 mg / L 1,2-hexanediol, 83.6 mg / L 1,2-heptanediol and 49.7 mg / L 1,2-octanediol yields. The method is simple to operate, green and environmentally friendly, suitable for large-scale industrial synthesis, and has good application prospects.

Claims

1. A biosynthetic method for a medium-chain 1,2-diol having a chain length of C6-C8, characterized in that: The steps include: A) Reduction of 2-oxocarboxylic acids to 2-hydroxycarboxylic acids; B) Reduction of 2-hydroxycarboxylic acids to 2-hydroxyaldehydes; C) Reduction of 2-hydroxyaldehydes to 1,2-diols; The chain length of the 2-oxocarboxylic acid is C6-C8.

2. The biosynthesis method according to claim 1, characterized in that: Step A) is to reduce 2-oxocarboxylic acid to 2-hydroxycarboxylic acid using 2-oxocarboxylic acid reductase; And / or, the method adopted in step B) is any one of the following methods: Method 1: 2-Hydroxycarboxylic acid is reduced to 2-hydroxyaldehyde by carboxylic acid reductase; or, Method 2: B1) 2-hydroxycarboxylic acid is converted to 2-hydroxyacyl-CoA by acyl CoA transferase or acyl CoA ligase; B2) 2-hydroxyacyl-CoA is reduced to 2-hydroxyaldehyde by acyl-CoA reductase; preferably, cinnamic acylase is also added to B1); And / or, step C) reduces the 2-hydroxyaldehyde to 1,2-diol using aldehyde reductase.

3. The biosynthesis method according to claim 2, characterized in that The biosynthesis method is a reduction and conversion under the conditions of at least one of whole cells, immobilized enzymes, a combination of purified enzymes, and crude enzyme solutions.

4. The biosynthesis method according to claim 3, characterized in that The whole cell is a recombinant microorganism; the starting strain of the recombinant microorganism is selected from Bacillus subtilis, Corynebacterium glutamicum, Yarrowia lipolytica, Saccharomyces cerevisiae or Escherichia coli.

5. The biosynthesis method according to claim 2, characterized in that: 2-oxocarboxylate reductase is encoded by one or more genes selected from the group consisting of ldh, ldhA, panE, ara, ara1, ara2, araDH, YGL039W, adh, CXQ87_003259; and / or, the carboxylate reductase is encoded by a CAR gene; and / or, the acyl-CoA transferase or acyl-CoA ligase is encoded by one or more genes selected from the group consisting of fldA, hadA, pct, Cat2, carB, 4CL, PaaK, Cat1, PGN_0725, PGN_1341, PGN_1888, fadD; and / or, the acyl-CoA reductase is encoded by one or more genes selected from the group consisting of maqu2220, maqu2507, bld, ald, pdcD, pduP, adhE, FAR, acr, hom, fadB, fadJ, hbd, phaB, phbB, mdh, ldh; and / or, the cinnamate acylase is encoded by one or more genes selected from the group consisting of: Pt4CL, At4CL2, At4CL5, Os4CL, Pc4CL; And / or, the aldehyde reductase is encoded by one or more genes selected from the group consisting of yqhD, ADH2, adh, bdh, yahK, adhA, 4-hbd, dhaT, mmsb, P84067, adhI, alrA, sucD, and bld.

6. The biosynthetic method according to any one of claims 1 to 5, characterized in that It also includes the following steps based on this biosynthesis method: increasing the intracellular precursor concentration or adding precursors in vitro such as threonine, pyruvate, cofactors NAD(P)H, NAD(P) + , ATP, etc. to further increase the production of medium-chain 1,2-diols with a chain length of C6-C8.

7. The biosynthetic method according to any one of claims 1 to 6, characterized in that: Taking a recombinant microorganism that overexpresses ldh, yqhD, fldA, maqu2507, and Pc4CL genes, culturing it in a medium containing cinnamic acid, inducing expression, adding 2-oxocarboxylic acid, and continuing fermentation to obtain 1,2-diol; Alternatively, a recombinant microorganism expressing the ilvA, ldh, zwf, yqhD, fldA, maqu2507, Pc4CL, and leuABCD genes is cultured in a medium containing cinnamic acid, expression is induced, threonine is added, and fermentation is continued to obtain 1,2-diol; Alternatively, a recombinant microorganism expressing cimA, ldh, zwf, yqhD, fldA, maqu2507, Pc4CL, and leuABCD genes is cultured in a medium comprising glucose and cinnamic acid, expression is induced, and fermentation is continued to obtain 1,2-diol.

8. The product obtained by the biosynthetic method according to any one of claims 1 to 7.

9. A recombinant microorganism, characterized in that The recombinant microorganism overexpresses genes for 2-oxocarboxylic acid reductase, carboxylic acid reductase, acyl-CoA reductase, acyl-CoA transferase, acyl-CoA ligase, cinnamate acylase and / or aldehyde reductase; Preferably, the starting strain is selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae or Yarrowia lipolytica.

10. The recombinant microorganism according to claim 9, characterized in that: 2-oxocarboxylate reductase is encoded by one or more genes selected from the group consisting of ldh, ldhA, panE, ara, ara1, ara2, araDH, YGL039W, adh, CXQ87_003259; and / or, the carboxylate reductase is encoded by a Car gene; and / or, the acyl-CoA reductase is encoded by one or more genes selected from the group consisting of maqu2220, maqu2507, bld, ald, pdcD, pduP, adhE, FAR, acr, hom, fadB, fadJ, hbd, phaB, phbB, mdh, ldh; and / or, the acyl-CoA transferase or acyl-CoA ligase is encoded by one or more genes selected from the group consisting of fldA, hadA, pct, Cat2, carB, 4CL, PaaK, Cat1, PGN_0725, PGN_1341, PGN_1888, fadD; and / or, the cinnamate acylase is encoded by one or more genes selected from the group consisting of: Pt4CL, At4CL2, At4CL5, Os4CL, Pc4CL; And / or, the aldehyde reductase is encoded by one or more genes selected from the group consisting of yqhD, ADH2, adh, bdh, yahK, adhA, 4-hbd, dhaT, mmsb, P84067, adhI, alrA, sucD, and bld.

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