Construction method of escherichia coli for efficiently synthesizing glycollic acid

Abstract

The invention discloses a construction method of escherichia coli for efficiently synthesizing glycollic acid, and belongs to the technical field of biochemical engineering. According to the invention, citric acid synthase gltA and aconitic acid synthase acnA and acnB are overexpressed in escherichia coli cells, pyruvate dehydrogenase transcription inhibition factor pdhR is knocked out, transhydrogenase pntA and pntB are overexpressed, and transhydrogenase stA is knocked out, so that the constructed recombinant escherichia coli can produce glycollic acid by using glucose in a short time. The reaction cost is lower, the yield is higher, the concentration of glycollic acid produced after fermentation in a 5L fermentation tank for 48 hours is 81 g / L, the conversion rate is 0.62 g / g, and the production intensity is 1.69 g / L / h.

Description

Technical Field

[0001] This invention relates to a method for constructing Escherichia coli that efficiently synthesizes glycolic acid, belonging to the field of biochemical technology. Background Technology

[0002] Glycolic acid (GA) is an α-hydroxy acid with the molecular formula C₂H₄O₃ and a relative molecular mass of 76.05. It is a widely used fine chemical product due to its structural characteristic of containing both hydroxyl and carboxyl groups. It is also an important intermediate in organic synthesis, with applications in chemical cleaning, daily chemical products, biodegradable materials, and pharmaceuticals. Currently, glycolic acid has a very wide range of uses, and with the progress of human society, its applications have even greater potential for development.

[0003] Glycolic acid is found in sugar beets, sugarcane, and unripe grape juice, but its content is very low and it is difficult to purify. Industrial production of glycolic acid uses chemical synthesis methods, such as the formaldehyde hydrocarboxylation method, which mainly uses formaldehyde, carbon monoxide, and water as raw materials; the chloroacetic acid method, which mainly uses chloroacetic acid, sodium hydroxide, and methanol as raw materials; and the cyanidation method, which mainly uses formaldehyde and hydrogen cyanide (or hydrofluoric acid) as raw materials. However, these processes are costly and cause serious environmental pollution. Meanwhile, researchers both domestically and internationally have conducted in-depth research on modifying microorganisms to produce glycolic acid using metabolic engineering techniques. For example, using *E. coli* as the producing strain, and employing rational metabolic engineering techniques and modular pathway engineering strategies, strain Mgly4340 was obtained with a glycolic acid yield of 65.5 g / L; another example is using *E. coli* ATCC8739 as the chassis strain to construct a synergistic system for the co-utilization of acetic acid and glucose, resulting in strain NZ-Gly303 with a glycolic acid yield of 73.3 g / L. However, all of the above strains are production strains containing plasmids. During industrial scale-up, factors such as plasmid loss, metabolic burden, inducers, and dependence on antibiotics can lead to unstable production, high costs, and safety risks.

[0004] Therefore, constructing a plasmid-free, resistance-free, and induction-free industrial production strain for glycolic acid is an urgent problem to be solved. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a recombinant Escherichia coli and its construction method and a method for efficiently synthesizing glycolic acid. The recombinant Escherichia coli of this invention can efficiently produce glycolic acid without the need for additional cofactor PLP, and has high production intensity, high conversion rate, and reduced production cost.

[0006] The first technical solution provided by this invention is to provide a recombinant Escherichia coli that efficiently synthesizes glycolic acid, overexpresses citrate synthase, aconitate synthase and transhydrogenase, and reduces the expression of transhydrogenase and pyruvate dehydrogenase transcriptional repressor factors.

[0007] In one embodiment, the recombinant Escherichia coli overexpresses citrate synthase gene gltA, aconitate synthase gene acnA, acnB and transhydrogenase gene pntA, pntB.

[0008] In one embodiment, the reduction of transaminase and pyruvate dehydrogenase transcriptional repressor expression is achieved by knocking out the transaminase gene sthA and the pyruvate dehydrogenase transcriptional repressor pdhR.

[0009] In one embodiment, the gene IDs of the genes for citrate synthase gltA, aconitate synthase acnA, acnB, transhydrogenase pntA, pntB, transhydrogenase sthA, and pyruvate dehydrogenase transcriptional repressor pdhR are 945323, 946724, 944864, 946628, 946144, 948461, and 944827, respectively. The nucleotide sequences of the citrate synthase gltA, aconitate synthase acnA, acnB, transhydrogenase pntA, and pntB are SEQ ID NO. 2-6.

[0010] In one embodiment, the citrate synthase gene gltA, aconitate synthase genes acnA and acnB, and transhydrogenase genes pntA and pntB are all generated by promoter P. trc Startup expression; the promoter P trc The nucleotide sequence is shown in SEQ ID NO1.

[0011] In one embodiment, the host of the recombinant Escherichia coli includes, but is not limited to, E. coli ATCC8739 or Escherichia coli GA-6; the Escherichia coli GA-6 is disclosed in patent application publication number CN120098871A. This strain is constructed by knocking out the lactate dehydrogenase gene ldhA, phosphoacetyltransferase gene pta, acetate kinase gene ackA, isocitrate dehydrogenase gene icd, malate synthase A gene aceB, malate synthase G gene glcB, aldehyde dehydrogenase gene aldA, and glycolate dehydrogenase gene glcDEF, and overexpressing isocitrate lyase aceA and glyoxylate reductase ghrA.

[0012] The present invention also provides a method for improving the glycolic acid production capacity of Escherichia coli, wherein the method involves overexpressing citrate synthase, aconitate synthase and transhydrogenase in Escherichia coli with glycolic acid synthesis capacity, and reducing the expression of transhydrogenase and pyruvate dehydrogenase transcriptional repressor.

[0013] In one embodiment, the citrate synthase gltA, aconitate synthase acnA, acnB, transhydrogenase pntA, and pntB are all generated by promoter P. trc The promoter expression, the promoter P trc The nucleotide sequence is shown in SEQ ID NO1.

[0014] The present invention also provides a method for preparing glycolic acid, wherein the recombinant Escherichia coli is fermented in a fermentation system to produce glycolic acid.

[0015] In one embodiment, the fermentation system contains phosphate, glucose, yeast extract, monosodium glutamate, and magnesium salt.

[0016] In one embodiment, the recombinant Escherichia coli is inoculated into the fermenter at an inoculation rate of 15%.

[0017] In one embodiment, during fermentation, glucose is fed in to control the residual sugar in the fermentation system to be between 0 g / L and 1 g / L, dissolved oxygen to be above 20%, 20% Ca(OH)2 is fed in to maintain the pH at 7.0 ± 0.2, the temperature is 37°C, and fermentation lasts for at least 48 h.

[0018] The present invention also provides the application of the recombinant Escherichia coli or the method in the production of glycolic acid or glycolic acid-containing products.

[0019] Technical effects: This invention provides a method for de novo production of glycolic acid via microbial fermentation. By enhancing the carbon flow of the glycolic acid synthesis pathway in *E. coli* cells (overexpressing citrate synthases gltA, aconitate synthases acnA and acnB, and knocking out the pyruvate dehydrogenase transcriptional repressor pdhR) and enhancing NADPH supply (overexpressing transhydrogenases pntA and pntB, and knocking out transhydrogenase sthA), the constructed recombinant *E. coli* can produce glycolic acid from glucose in a short time. This reaction has lower cost and higher yield; after 48 hours in a 5 L fermenter, the glycolic acid concentration produced is 81 g / L, the conversion rate is 0.57 g / g, and the production intensity is 1.69 g / L / h. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the metabolic pathway of the recombinant strain.

[0021] Figure 2The results of the fed-batch fermentation of strain GA-10 constructed in this invention in a 5L fermenter are shown. Detailed Implementation

[0022] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The solutions involved in the present invention are as follows: The culture media involved in the examples are as follows: LB medium: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone.

[0023] Primary seed culture medium: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 1 g / L sodium glutamate.

[0024] Secondary seed culture medium: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 1 g / L sodium glutamate, 1 g / L glucose.

[0025] Fermentation medium: 2.4 g / L potassium dihydrogen phosphate, 5.6 g / L dipotassium hydrogen phosphate, 15 g / L glucose, 7.5 g / L yeast extract, 5 g / L sodium glutamate, 0.5 g / L magnesium sulfate heptahydrate.

[0026] The plasmids, strains, and primers involved in the examples are shown in Tables 1-3.

[0027] Table 1 shows plasmid information.

[0028] Table 2 shows the strain information.

[0029] Table 3 Primers and Sequences

[0030] Perform PCR amplification according to the following PCR amplification system.

[0031] Table 4 PCR amplification system

[0032] Table 5 PCR amplification conditions

[0033] Example 1: Construction of an engineered strain GA-7 with the gltA gene integrated into its genome. by E.coli Starting with GA-6 (published in patent application CN120098871A), an engineered strain GA-7 was constructed by integrating the citrate synthase gltA gene into its genome. Its promoter P... trc The nucleotide sequence is shown in SEQ ID NO. 1. All gene editing operations were performed using CRISPR-Cas9 gene editing technology.

[0034] The specific method is as follows: (1) with E.coli Using the genome of strain ATCC8739 as a template, primers were used... ygaY-up500-F and ygaY- up500-R , ygaY-down500-F and ygaY-down500-R Amplification ygaY Homologous arms above and below the locus (Gene ID: 4056025), using primers gltA-F and gltA-R Amplification gltA Gene sequence (nucleotide sequence as shown in SEQ ID NO.2), then using primers ygaY-up500-F and ygaY-down500-R upper and lower arms and gltA Gene fusion ygaY :: gltA Integration box; (2) Using pTarget plasmid as a template ygaY-n20-F and ygaY-n20-R Using primers, linearized fragments were amplified, and homologous recombination was performed to transform the cloning host. E.coli DH5α From the middle, extract pTarget-ygaY plasmids; (3) Verify the correct pTarget-ygaY plasmids and ygaY :: gltA The integrative frame was electroporated into GA-6 containing the pCas9 plasmid. After electroporation, it was plated on LB plates containing kanamycin and spectinomycin and cultured at 30°C. (4) Use validation primers ygaY-up500-F and ygaY-up500-R Colony PCR was performed, and the correct transformants were obtained by sequencing. (5) Transfer the correct transformants to LB medium containing 0.1% IPTG and culture at 30°C to eliminate pTarget plasmid. Then, streak the plates to select the correct transformants. (6) Transfer the correct transformants to LB medium and culture at 42°C to eliminate pCas9 plasmid. Then, streak the plates to screen for the correct transformants. (7) Obtain E.coli GA-7.

[0035] Example 2: Construction of engineered strain GA-8 with integrated acnA and acnB genes in its genome Constructed using Example 1 E.coli GA-7 was the starting strain, which integrated aconitine synthase. acnA , acnB Its promoter P trc The nucleotide sequence is shown in SEQ ID NO.1, and the engineered strain is constructed. E.coli All gene editing operations in GA-8 were performed using CRISPR-Cas9 gene editing technology.

[0036] The specific method is as follows: (1) with E.coli Using the genome of strain ATCC8739 as a template, primers were used... ylbE-down500-F and ylbE- up500-R , ylbE-down500-F and ylbE-down500-R Amplification ygaY Homologous arms above and below the locus (Gene ID: 2847696), using primers acnA-F and acnA-R Amplification acnA Gene sequence (nucleotide sequence as shown in SEQ ID NO.3), then using primers ylbE-up500-F and ylbE-down500-R upper and lower arms and acnA Gene fusion ylbE :: acnA Integration box; (2) Using pTarget plasmid as a template ylbE-n20-F and ylbE-n20-R Using primers, linearized fragments were amplified, and homologous recombination was performed to transform the cloning host. E.coli DH5α From the middle, extract pTarget-ylbE plasmids; (3) Verify the correct pTarget-ylbE plasmids and ylbE :: acnA The integrative frame was electroporated into GA-7 containing the pCas9 plasmid. After electroporation, it was plated on LB plates containing kanamycin and spectinomycin and cultured at 30°C. (4) Use validation primers ylbE-up500-F and ylbE-up500-R Colony PCR was performed, and the correct transformants were obtained by sequencing. (5) Transfer the correct transformants to LB medium containing 0.1% IPTG and culture at 30°C to eliminate pTarget plasmid. Then, streak the plates to select the correct transformants. (6) Transfer the correct transformants to LB medium and culture at 42°C to eliminate pCas9 plasmid. Then, streak the plates to screen for the correct transformants. (7) Same as the steps above. yeeL Locus (Gene ID: 2847764) integration acnB The gene (nucleotide sequence as shown in SEQ ID NO.4) was obtained. E.coli GA-7.

[0037] Example 3: Constructing a genome knockout sthA Genes, Integration pntAB Gene-engineered strain GA-9 Constructed using Example 2 E.coli Using GA-8 as the starting strain, a genome-knockout transhydrogenase was constructed. sthA Gene (Gene ID: 948461), Integrase transhydrogenase pntAB The engineered strain GA-9, whose promoter is P trc The nucleotide sequence is shown in SEQ ID NO.1. All gene editing operations were performed using CRISPR-Cas9 gene editing technology.

[0038] The specific method is as follows: (1) with E.coli Using the genome of strain ATCC8739 as a template, primers were used... sthA-up500-F and sthA- up500-R , sthA-down500-F and sthA-down500-R Amplification sthA Homologous arms above and below the site, using primers pntAB-F and pntAB-R Amplification pntAB Gene sequence ( pntA , pntB The nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively, and then primers are used. sthA-up500-F and sthA-down500-R upper and lower arms and pntAB Gene fusion sthA :: pntAB Integration box; (2) Using pTarget plasmid as a template sthA-n20-F and sthA-n20-R Using primers, linearized fragments were amplified, and homologous recombination was performed to transform the cloning host. E.coli DH5α From the middle, extract pTarget-sthA plasmids; (3) Verify the correct pTarget-sthA plasmids and sthA :: pntAB The integrative frame was electroporated into GA-8 containing the pCas9 plasmid. After electroporation, it was plated on LB plates containing kanamycin and spectinomycin and cultured at 30°C. (4) Use validation primers sthA-up500-F and sthA-up500-R Colony PCR was performed, and the correct transformants were obtained by sequencing. (5) Transfer the correct transformants to LB medium containing 0.1% IPTG and culture at 30°C to eliminate pTarget plasmid. Then, streak the plates to select the correct transformants. (6) Transfer the correct transformants to LB medium and culture at 42°C to eliminate pCas9 plasmid. Then, streak the plates to screen for the correct transformants. (7) Obtain E.coli GA-9.

[0039] Example 4: Constructing a genome knockout pdhR Genetically engineered strain GA-10 Constructed using Example 3 E.coli Using GA-9 as the starting strain, a genome-knockout transhydrogenase was constructed. pdhR The engineered strain GA-9, with gene (Gene ID: 944827), has a promoter P trc The nucleotide sequence is shown in SEQ ID NO.1. All gene editing operations were performed using CRISPR-Cas9 gene editing technology.

[0040] The specific method is as follows: (1) with E.coli Using the genome of strain ATCC8739 as a template, primers were used... pdhR-up500-F and ​ ​ , ​ and ​ Amplification ​ Homologous arms above and below the site, then use primers. ​ ​ and ​ Merge the upper and lower arms into ​ Knockout box; (2) Using pTarget plasmid as a template ​ and ​ Using primers, linearized fragments were amplified, and homologous recombination was performed to transform the cloning host. ​ From the middle, extract ​ plasmids; (3) Verify the correct ​ plasmids and​ The knockout frame was electroporated into GA-9 containing the pCas9 plasmid. After electroporation, the plasmid was plated on LB plates containing kanamycin and spectinomycin and cultured at 30°C. (4) Use validation primers ​ and ​ Colony PCR was performed, and the correct transformants were obtained by sequencing. (5) Transfer the correct transformants to LB medium containing 0.1% IPTG and culture at 30°C to eliminate pTarget plasmid. Then, streak the plates to select the correct transformants. (6) Transfer the correct transformants to LB medium and culture at 42°C to eliminate pCas9 plasmid. Then, streak the plates to screen for the correct transformants. (7) Obtain ​ GA-10.

[0041] Example 5: Recombinant strain ​ GA-10 was fed-batch fermented in a 5 L fermenter. The recombinant strain prepared in Example 3 was prepared according to the following loading process. ​ GA-9, the recombinant strain prepared in Example 4 ​ GA-10 is used in 5 L fermenters for fed-batch fermentation. Details are as follows: (1) Slant activation: The bacterial cells in the glycerol tube were streaked onto the slant culture medium and incubated at 37°C for 20-24 h.

[0042] (2) Primary shake-flask seed culture medium: Wash the colonies off the slant culture medium with sterile water, and inoculate them at a 2% inoculation rate into a 500 mL Erlenmeyer flask containing 100 mL of culture medium. Incubate at 37°C for 6-8 h at 200 rpm. OD 600 Between 6.0 and 10.0.

[0043] (3) Secondary shake-flask seed culture medium: Inoculate the primary seed culture at a rate of 2% into a 500 mL Erlenmeyer flask containing 100 mL of secondary seed culture medium. Incubate at 200 rpm and 37°C for 6–8 h. Add 100 μL of 50% ammonia solution at 4.5 h. OD 600 Between 8.0 and 1.0; (4) 5 L Dubil parallel reactor fermentation: The initial liquid volume of the fermenter is 2.5 L. The secondary seed liquid is inoculated into the fermentation medium at an inoculation rate of 10% (volume ratio) of the total volume. The temperature is 37°C, the initial aeration rate is 1.5 vvm, glucose is added during fermentation to control the residual sugar of the fermentation system at 0 g / L-1 g / L, Ca(OH)2 is added during fermentation to control the pH at 7.0±0.2, the dissolved oxygen is controlled to be not less than 20%, and the fermentation time is at least 48 h. Fermentation medium: 2.4 g / L potassium dihydrogen phosphate, 5.6 g / L dipotassium hydrogen phosphate, 15 g / L glucose, 7.5 g / L yeast extract, 5 g / L sodium glutamate, 0.5 g / L magnesium sulfate heptahydrate, 1 mL / L antifoaming agent.

[0044] The glycolic acid yield was measured after 48 h of fermentation. The results showed that strain GA-10 achieved a glycolic acid yield of 81.0 g / L, with a yield and production intensity of 0.62 g / g and 1.69 g / L / h, respectively. Furthermore, the production performance of strain GA-9 was also tested, with a glycolic acid yield, yield, and production intensity of 76.5 g / L, 0.61 g / g, and 1.59 g / L / h, respectively.

[0045] Comparative Example 1: The recombinant bacteria E. coli GA-7 to E. coli GA-10 constructed in Examples 1-4 were fermented in a 5L fermenter system for 48 h according to the method in Example 5. The glycolic acid yield in the fermentation broth was detected. The results showed that the yields of strains E. coli GA-7 to E. coli GA-10 were 57.9 g / L, 62.5 g / L, 76.5 g / L and 81.0 g / L, respectively, with yields of 0.59 g / g, 0.61 g / g, 0.61 g / g and 0.62 g / g, respectively, and production intensities of 1.19 g / L / h, 1.27 g / h, 1.59 g / L / h and 1.69 g / L / h, respectively.

[0046] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant Escherichia coli for efficient synthesis of glycolic acid, characterized by, Overexpressing citrate synthase, aconitate synthase and transhydrogenase, and reducing the expression of transhydrogenase and pyruvate dehydrogenase transcriptional repressor.

2. The recombinant E. coli of claim 1, wherein, The recombinant E. coli overexpresses citrate synthase gene gltA, aconitate synthase genes acnA, acnB and transhydrogenase genes pntA, pntB.

3. The recombinant E. coli according to claim 1 or 2, characterized in that, Knocking out transhydrogenase gene sthA and pyruvate dehydrogenase transcriptional repressor pdhR.

4. The recombinant E. coli of any one of claims 1-3, wherein, The citrate synthase gene gltA, aconitate synthase genes acnA, acnB, transhydrogenase genes pntA, pntB are under the control of the promoter P trc initiate expression.

5. The recombinant E. coli of any one of claims 1-4, wherein, The host includes but is not limited to E. coli ATCC8739 or E. coli GA-6.

6. A method of increasing the production capacity of glycolic acid by E. coli, characterized by, Overexpressing citrate synthase, aconitate synthase and transhydrogenase, and reducing the expression of transhydrogenase and pyruvate dehydrogenase transcriptional repressor in E. coli with glycolate synthesis ability.

7. A process for the preparation of glycolic acid, characterized in that, The recombinant E. coli of any one of claims 1-5 is fermented in a fermentation system with glucose as carbon source to produce glycolic acid.

8. The method of claim 7, wherein, During the fermentation process, the glucose is fed to control the glucose in the fermentation system not more than 1 g / L, the dissolved oxygen is controlled at more than 20%, the pH is maintained at 6.8-7.2, the temperature is maintained at 35-40°C, and the fermentation is maintained for at least 48 h.

9. The method according to claim 7 or 8, characterized in that, The fermentation system contains phosphate, glucose, yeast extract, sodium glutamate and magnesium salt.

10. Use of the recombinant E. coli of any one of claims 1-5 or the method of any one of claims 6-9 in the production of glycolic acid or glycolic acid-containing products.

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