CN116083329A - Method for producing gamma-butyrolactone or 1,4-butanediol by fermentation - Google Patents

Abstract

The invention provides a method for fermenting and producing γ-butyrolactone or 1,4-butanediol. Through synthetic biology technology, Corynebacterium glutamicum is used as a chassis, and an exogenous glutamic acid decarboxylase gene gadB is introduced. * Mutant, alcohol dehydrogenase gene yqhD, enhanced endogenous or exogenous γ-aminobutyric acid transaminase gene gabT, realized the production of 4-hydroxybutyric acid by one-step fermentation of renewable raw materials such as glucose and sucrose by recombinant microorganisms , and then decarboxylate the fermentation broth by simple heating to obtain GBL. Further introducing the carboxylic acid reductase CAR gene and the phosphopantetheinyl transferase gene sfp into the above-mentioned microorganisms can realize the direct fermentation of glucose, sucrose and the like to produce BDO by using the recombinant microorganisms. The invention realizes the direct fermentation production of important chemicals GBL and BDO by using renewable raw materials, the production process is clean and safe, the carbon emission is low, and has important industrial application value.

Description

Method for producing gamma-butyrolactone or 1,4-butanediol by fermentation

technical field

The invention belongs to the technical field of genetic engineering and biological fermentation, and in particular relates to a method for producing gamma-butyrolactone or 1,4-butanediol by fermentation.

Background technique

1,4-Butanediol (BDO) is an important organic chemical and fine chemical raw material, which is widely used in the manufacture of various polymer materials such as polyester, polyurethane and polyether polyols. For example, it is a synthetic engineering plastic polymer It is the key monomer of butylene terephthalate (PBT), and it is also the key monomer of synthesizing biodegradable materials polybutylene succinate (PBS) and PBAT. In recent years, with the introduction of plastic restrictions and carbon neutral policies, the demand for biodegradable materials PBS and PBAT has shown a blowout development, which has driven the rapid growth of the global market demand for BDO. BDO is also an important precursor for the synthesis of tetrahydrofuran, γ-butyrolactone and other chemicals. γ-butyrolactone (GBL) is also an important organic and pharmaceutical intermediate, which is widely used in medicine, pesticide, petrochemical industry, etc. It is one of the main components of lithium battery electrolyte, and it is also a -Important raw materials for chemicals such as methyl-2-pyrrolidone (NMP) and polyvinylpyrrolidone (PVP).

At present, the synthesis of BDO is mainly through chemical methods, using calcium carbide as raw materials through a complex chemical conversion process, which is an extremely high-energy-consuming process. The direct fermentation of biomass raw materials to produce BDO has important industrial application value. The production of GBL is currently mainly based on BDO as a raw material through chemical production, and there is no relevant report on the direct synthesis of GBL by biological methods.

Contents of the invention

The object of the present invention is to provide a method for fermentatively producing γ-butyrolactone or 1,4-butanediol.

The concept of the present invention is as follows: through synthetic biology technology, using Corynebacterium glutamicum as the chassis, introducing exogenous glutamic acid decarboxylase gene gadB* mutant, alcohol dehydrogenase gene yqhD, and strengthening endogenous Or the exogenous γ-aminobutyric acid transaminase gene gabT, for the first time realized the use of recombinant microorganisms to ferment renewable raw materials such as glucose and sucrose to produce 4-hydroxybutyric acid in one step, and then simply heat and dehydrate the fermentation broth to obtain GBL . Further introducing the carboxylic acid reductase CAR gene and the phosphopantetheinyl transferase gene sfp into the above-mentioned microorganisms can realize the direct fermentation of glucose, sucrose and the like to produce BDO by using the recombinant microorganisms.

In order to realize the object of the present invention, the first aspect, the present invention provides a kind of recombinant Corynebacterium glutamicum, described recombinant Corynebacterium glutamicum is by overexpressing glutamic acid decarboxylase mutant gene gadB in Corynebacterium glutamicum *Constructed with alcohol dehydrogenase gene yqhD.

In the present invention, the glutamic acid decarboxylase mutant gene gadB* and the alcohol dehydrogenase gene yqhD are derived from Escherichia coli; the amino acid sequences encoded by the gene gadB* and the gene yqhD are shown in SEQ ID NO: 1 and 2, respectively.

Further, the endogenous or exogenous γ-aminobutyric acid transaminase gene gabT is expressed in the recombinant Corynebacterium glutamicum.

The gamma-aminobutyric acid transaminase gene gabT can be derived from Corynebacterium glutamicum, Escherichia coli or Pseudomonas putida, and the amino acid sequences encoded by them are respectively as SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO :5 shown.

In the second aspect, the present invention provides the application of the recombinant Corynebacterium glutamicum in fermentative production of γ-butyrolactone.

Further, the recombinant Corynebacterium glutamicum is used to ferment and produce γ-butyrolactone with cheap carbon source as raw material.

The cheap carbon source can be selected from at least one of glucose, sucrose, maltose, cellobiose and the like.

In a third aspect, the present invention provides a Corynebacterium glutamicum engineering bacterium, and the engineering bacterium expresses the carboxylic acid reductase CAR gene and the phosphopantetheinyl transferase gene in the recombinant Corynebacterium glutamicum sfp build got.

The carboxylic acid reductase CAR gene can be derived from Mycobacterium marinum, Nocardiaiowensis, Mycolicibacterium smegmatis or Mycobacteroides abscessus, and the amino acid sequences encoded by them are respectively as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO : as shown in 9.

The phosphopantetheinyl transferase gene sfp can be derived from Bacillus subtilis, and its encoded amino acid sequence is shown in SEQ ID NO:10.

In the fourth aspect, the present invention provides the application of the Corynebacterium glutamicum engineering bacteria in the fermentative production of 1,4-butanediol.

Further, the Corynebacterium glutamicum engineering bacteria are used to ferment and produce γ-butyrolactone with cheap carbon sources as raw materials.

The cheap carbon source can be selected from at least one of glucose, sucrose, maltose, cellobiose and the like.

In a fifth aspect, the present invention provides a glutamic acid decarboxylase mutant whose amino acid sequence is shown in SEQ ID NO:1.

In the sixth aspect, the present invention provides a method for constructing the recombinant Corynebacterium glutamicum and the Corynebacterium glutamicum engineering bacterium, and the above-mentioned genes can be transformed or modified by conventional genetic engineering methods.

By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

The present invention mainly adopts synthetic biology technology, uses Corynebacterium glutamicum as the chassis, and introduces exogenous glutamic acid decarboxylase gene gadB* mutant, alcohol dehydrogenase gene yqhD into the chassis, and strengthens endogenous or exogenous The original γ-aminobutyric acid transaminase gene gabT has pioneered the use of recombinant microorganisms to ferment renewable raw materials such as glucose and sucrose to produce 4-hydroxybutyric acid in one step, and then simply heat and decarboxylate the fermentation broth to obtain GBL . Further introducing the carboxylic acid reductase CAR gene and the phosphopantetheinyl transferase gene sfp into the above-mentioned microorganisms can realize the direct fermentation of glucose, sucrose and the like to produce BDO by using the recombinant microorganisms. The invention realizes the direct fermentation production of important chemicals GBL and BDO by using renewable raw materials, the production process is clean and safe, and the carbon emission is reduced by more than 60% compared with the chemical method, and has important industrial application value.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Embodiment 1 produces the construction method of the recombinant Corynebacterium glutamicum of GBL

There are no microorganisms in nature that can synthesize GBL naturally. In the present invention, by introducing glutamic acid decarboxylase gabB* into Corynebacterium glutamicum, the glutamic acid produced by Corynebacterium glutamicum is first converted into γ-aminobutyric acid, and the latter is converted into γ-aminobutyric acid by itself or exogenously introduced γ-aminobutyric acid. -Aminobutyric acid transaminase gabT and exogenously introduced alcohol dehydrogenase yqhD are further converted into 4-hydroxybutyric acid, and 4-hydroxybutyric acid in the fermentation broth produces GBL under acid catalysis.

The gene fragment of glutamic acid decarboxylase gabB* of Escherichia coli is artificially synthesized (the amino acid sequence is shown in SEQ ID NO: 1, and the gene sequence is shown in SEQ ID NO: 11). Compared with wild-type glutamic acid decarboxylase, the glutamic acid decarboxylase has higher activity under neutral pH conditions. Using the gene fragment as a template, PCR was performed with gabB-F (5'-attaagcttgcatgcctgcactttaagaaggagatataccatggataagaagcaagtaacg-3') and gabB-R (5'-ggtatatctccttcttaaagttagtgatcgctgagatatt-3') primers to obtain a gabB fragment of about 1.4 kb and perform PCR purification. Using the genome of Escherichia coli MG1655 as a template, PCR was performed with yqhD-F (5′-ctttaagaaggagatataccatgaacaactttaatctgcac-3′) and yqhD-R (5′-ggtacccggggatcctctagttagcgggcggcttcgtata-3′) as primers to obtain a yqhD fragment of about 1.2 kb and perform PCR purification. The pXMJ19 plasmid (purchased from Addgene) was double digested with PstI and XbaI, and the gabB fragment and yqhD fragment obtained above were ligated to pXMJ19 in one step using the Gibson Assembly kit (NEB), and the obtained recombinant plasmid was named pXMJ-gabB -yqhD.

Using the genome of Corynebacterium glutamicum as a template, PCR was performed with gabT_cg-F (5′-ggatccccgggtaccgagctctttaagaaggagatataccgtggaagatctctcataccg-3′) and gabT_cg-R (5′-caaaacagccaagctgaattttagcccaccttctggtgcg-3′) as primers to obtain a gab of about 1.35 kb T_cg fragment and Perform PCR purification. Using the genome of Escherichia coli MG1655 as a template, PCR was performed with gabT_ec-F (5′-ggatccccgggtaccgagctctttaagaaggagatataccatgaacagcaataaagagtt-3′) and gabT_ec-R (5′-caaaacagccaagctgaattctactgcttcgcctcatcaa-3′) as primers to obtain about 1.35kb gab T_ec fragment and perform PCR purification. Using the genome of Pseudomonas putida as a template, the primers gabT_pp-F (5′-ggatccccgggtaccgagctctttaagaaggagatataccatgagcaagaccaacgaatc-3′) and gabT_pp-R (5′-caaaacagccaagctgaatttcaggcaagttcagcgaagc-3′) were used as primers to perform PCR to obtain about 1.35kb gab T_pp fragment And perform PCR purification. The plasmid pXMJ-gabB-yqhD was double digested with KpnI and EcoRI, and the gabT_cg fragment, gabT_ec fragment, and gabT_pp fragment obtained from the above purification were respectively connected to pXMJ-gabB-yqhD using the Gibson Assembly kit (NEB), and the obtained recombinant The plasmids were named pXMJ-gabB-yqhD-gabT_cg, pXMJ-gabB-yqhD-gabT_ec and pXMJ-gabB-yqhD-gabT_pp, respectively.

Plasmids pXMJ-gabB-yqhD, pXMJ-gabB-yqhD-gabT_cg, pXMJ-gabB-yqhD-gabT_ec, and pXMJ-gabB-yqhD-gabT_pp were transferred into Corynebacterium glutamicum S9114 by electroporation, in the presence of 10 mg/ The recombinant bacteria were screened on the chloramphenicol LB plate of L and named as S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec and S9114/pXMJ-gabB- yqhD-gabT_pp. At the same time, the pXMJ19 empty plasmid was also transformed into Corynebacterium glutamicum S9114 to obtain the control strain S9114/pXMJ.

Example 2 Production of GBL by Fermentation of Cheap Sugar Raw Materials

The strain S9114/PXMJ-Gabb-YQHD, S9114/PXMJ-Gabb-Yqhd-Gabt_cg, S9114/PXMJ-Gabb-Yqhd-Gabt_EC, S9114/PXMJ-Gabb-Gabt_pp and the control strain S9114 /pxmj to inoculate to 5L fermentation tank The initial volume of the fermentation broth is 2L, the fermentation temperature is 30°C, and the ventilation volume is 1vvm. During the fermentation process, the dissolved oxygen value in the fermentation process is maintained at 20% by adjusting the rotation speed, and the fermentation broth is controlled by automatically adding 25% ammonia water. The pH was 7.0, and 0.1mM IPTG was added for induction after 4 hours of fermentation, and the fermentation time was 48 hours.

Fermentation medium formula includes (g/L): glucose 100, (NH ₄ ) ₂ SO ₄ 20, K ₂ HPO ₄ 1.0, MgSO ₄ 0.5, MnSO ₄ 0.2, FeSO ₄ 0.2, corn steep liquor 20, pyridoxal phosphate 0.01 , thiamine hydrochloride 0.001, chloramphenicol 0.005.

Stop the fermentation after 48 hours of fermentation, add concentrated HCl to the fermentation broth to adjust the pH value to 0.8, react at room temperature for 24 hours, and then use high performance liquid chromatography (HPLC) to detect the product of the strain. Strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp can produce 7.2g/L and 11.3g/L, respectively , 14.1g/L, 13.7g/L of GBL, while the control strain S9114/pXMJ did not produce GBL. It shows that the introduction of artificial pathway in Corynebacterium glutamicum can successfully realize the efficient production of glucose to GBL.

The carbon source of the fermentation medium was changed from 100g/L glucose to 100g/L sucrose, other components remained unchanged, and the fermentation process remained completely the same. Strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp can produce 7.4g/L and 10.2g/L, respectively , 13.3g/L, 12.9g/L of GBL, while the control strain S9114/pXMJ did not produce GBL. It shows that the introduction of artificial pathways in Corynebacterium glutamicum can successfully achieve the efficient production of sucrose to GBL.

The carbon source of the fermentation medium is changed from 100g/L glucose to molasses (wherein the sucrose content is 100g/L), other components remain unchanged, and the fermentation process also remains completely consistent. Strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp can produce 6.7g/L and 9.9g/L, respectively , 12.1g/L, 11.7g/L of GBL, while the control strain S9114/pXMJ did not produce GBL. It shows that the introduction of artificial pathways in Corynebacterium glutamicum can successfully achieve the efficient production of molasses to GBL.

Embodiment 3 produces the construction method of the recombinant Corynebacterium glutamicum of BDO

The strain constructed above can efficiently transform cheap carbon sources such as glucose, sucrose, and molasses to produce GBL. The invention further converts 4-hydroxybutyric acid, the precursor of GBL, into BDO by introducing carboxylic acid reductase, so as to realize the direct fermentation and production of BDO by using cheap carbon sources.

Artificially synthesized phosphopantetheinyl transferase gene sfp of Bacillus subtilis (the amino acid sequence is shown in SEQ ID NO: 10, and the gene sequence is shown in SEQ ID NO: 20), the main function of the enzyme is to activate carboxylic acid reduction enzyme. Using the gene fragment as a template, PCR was performed with primers sfp-F (5′-atcctctagagtcgacctgcactttaagaaggagatataccaatgaaaatttacggcatcta-3′) and sfp-R (5′-cagtgccaagcttgcatgcctcaaagtaactcctcgtagg-3′) to obtain a sfp fragment of about 0.7 kb and perform PCR purification. The pEC-K18mob plasmid (purchased from Addgene) was single-digested with PstI, and the sfp fragment obtained above was ligated to pEC-K18mob in one step using the Gibson Assembly kit (NEB), and the obtained recombinant plasmid was named pEC-sfp.

Carboxylate reductase genes Car_mm, Car_ni, Car_ms, and Car_ma derived from Mycobacterium marinum, Nocardia iowensis, Mycolicibacterium smegmatis, and Mycobacteroides abscessus (amino acid sequences are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively) , SEQ ID NO: 9, the nucleotide sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 respectively). The plasmid pEC-sfp was double digested with EcoRI and KpnI, and the four gene fragments synthesized above were respectively inserted into the plasmid pEC-sfp, and the obtained plasmids were named pEC-Car_mm-sfp, pEC-Car_ni-sfp, pEC -Car_ms-sfp, pEC-Car_ma-sfp. The plasmids pEC-Car_mm-sfp, pEC-Car_ni-sfp, pEC-Car_ms-sfp, and pEC-Car_ma-sfp were transformed into Corynebacterium glutamicum strain S9114/pXMJ-gabB-yqhD-gabT_ec respectively, and the obtained recombinant strains were named respectively For S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA.

Bacterial strains S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA and control bacterial strains S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ were inoculated into a 5L fermenter for cultivation, The initial volume of the fermentation broth is 2L, the fermentation temperature is 30°C, and the ventilation rate is 1vvm. During the fermentation process, the dissolved oxygen value during the fermentation process is maintained at 20% by adjusting the rotation speed, and the pH of the fermentation broth is controlled to 7.0 by automatically adding 25% ammonia water. , adding 0.1m MIPTG for induction at 4 hours of fermentation, and the fermentation time was 48 hours.

Fermentation medium formula includes (g/L): glucose 100, (NH ₄ ) ₂ SO ₄ 20, K ₂ HPO ₄ 1.0, MgSO ₄ 0.5, MnSO ₄ 0.2, FeSO ₄ 0.2, corn steep liquor 20, pyridoxal phosphate 0.01 , Thiamine hydrochloride 0.001, chloramphenicol 0.005.

After 48 hours of fermentation, the fermentation was stopped, and the products of the strains were detected by high performance liquid chromatography (HPLC). Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, and S9114-X-MA could produce 7.8g/L, 6.9g/L, 7.2g/L, and 7.7g/L of BDO, respectively, while The control strains S9114/pXMJ-gabB-yqhD-gabT_ec and S9114/pXMJ did not produce BDO. It shows that the introduction of artificial pathway in Corynebacterium glutamicum can successfully realize the efficient production of glucose to BDO.

The carbon source of the fermentation medium was changed from 100g/L glucose to 100g/L sucrose, other components remained unchanged, and the fermentation process remained completely the same. Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, and S9114-X-MA could produce 6.4g/L, 6.2g/L, 6.3g/L, and 6.9g/L of BDO, respectively, while The control strains S9114/pXMJ-gabB-yqhD-gabT_ec and S9114/pXMJ did not produce BDO. It shows that the introduction of artificial pathway in Corynebacterium glutamicum can successfully realize the efficient production of sucrose to BDO.

The carbon source of the fermentation medium is changed from 100g/L glucose to molasses (wherein the sucrose content is 100g/L), other components remain unchanged, and the fermentation process also remains completely consistent. Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, and S9114-X-MA could produce 5.7g/L, 5.9g/L, 5.1g/L, and 4.7g/L of BDO, respectively, while The control strains S9114/pXMJ-gabB-yqhD-gabT_ec and S9114/pXMJ did not produce BDO. It shows that the introduction of artificial pathway in Corynebacterium glutamicum can successfully realize the efficient production of molasses to BDO.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. Recombinant Corynebacterium glutamicum, characterized in that, said recombinant Corynebacterium glutamicum is constructed by overexpressing glutamic acid decarboxylase mutant gene gadB* and alcohol dehydrogenase gene yqhD in Corynebacterium glutamicum owned; The glutamic acid decarboxylase mutant gene gadB* and the alcohol dehydrogenase gene yqhD are derived from Escherichia coli; the amino acid sequences encoded by the gene gadB* and the gene yqhD are shown in SEQ ID NO: 1 and 2, respectively. 2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that, the endogenous or exogenous γ-aminobutyric acid transaminase gene gabT is further expressed in the recombinant Corynebacterium glutamicum. 3. recombinant Corynebacterium glutamicum according to claim 2, is characterized in that, described gamma-aminobutyric acid transaminase gene gabT is derived from Corynebacterium glutamicum, Escherichia coli or Pseudomonas putida, and their encoded The amino acid sequences are respectively shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. 4. The application of the recombinant Corynebacterium glutamicum described in any one of claims 1-3 in the fermentative production of γ-butyrolactone. 5. The application according to claim 4, characterized in that, fermenting and producing gamma-butyrolactone from a cheap carbon source; The cheap carbon source is at least one selected from glucose, sucrose, maltose and cellobiose. 6. Corynebacterium glutamicum engineering bacterium, it is characterized in that, described engineering bacterium expresses carboxylic acid reductase CAR gene and phosphopantethein in the recombinant Corynebacterium glutamicum described in any one of claim 1-3 Aminotransferase gene sfp was constructed. 7. The engineering bacterium according to claim 6, wherein the carboxylic acid reductase CAR gene is derived from Mycobacterium marinum, Nocardia iowensis, Mycolicibacterium smegmatis or Mycobacteroides abscessus, and the amino acid sequences encoded by them are respectively as SEQ ID NO:6 , SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; and/or The phosphopantetheinyl transferase gene sfp is derived from Bacillus subtilis, and its encoded amino acid sequence is shown in SEQ ID NO:10. 8. the application of the engineering bacterium described in claim 6 or 7 in fermentative production of 1,4-butanediol. 9. The application according to claim 8, characterized in that 1,4-butanediol is fermented from a cheap carbon source as a raw material; The cheap carbon source is at least one selected from glucose, sucrose, maltose and cellobiose. 10. Glutamic acid decarboxylase mutant, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:1.