Genetically engineered bacterium and method for producing succinic acid by fermenting corn straw hydrolysate

Abstract

The invention discloses a genetically engineered bacterium and method for producing succinic acid by fermenting corn straw hydrolysate, and the method comprises the following specific steps: 1, preparing the corn straw hydrolysate: treating 1-2% NaOH solution at 110-130 DEG C for 0.5-1.5 h, with the lignin removal rate being greater than or equal to 70% and the cellulose retention rate being greater than or equal to 85%; step 2, aerobic fermentation of the genetically engineered bacteria: a culture medium contains 8-12 g / L of glucose and 3-7 g / L of yeast extract or corn steep liquor, and the genetically engineered bacteria are cultured at 35-39 DEG C and 180-220 rpm until OD600 is 2.5-3.5; and 3, anaerobic fermentation of the bacterial strain: taking the corn straw hydrolysate obtained in the step 1 as a carbon source, and adding a buffering agent into the genetically engineered bacteria for anaerobic fermentation. According to the recombinant escherichia coli E.coli SUCC-01, succinic acid is produced by knocking out an acetic acid synthesis gene pta / ackA and overexpressing an sdhA / mdh gene and combining with corn straw hydrolysate through fermentation, the yield of the succinic acid can reach 80 g / L, the total sugar utilization rate is larger than or equal to 90%, the by-product acetic acid is smaller than or equal to 12 g / L, and the yield is increased by 60% compared with that of a wild type strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering and fermentation engineering, and specifically relates to a genetically engineered bacterium for efficiently fermenting succinic acid using corn straw hydrolyzate through genetic engineering of Escherichia coli and a preparation method thereof. Background Art

[0002] Succinic acid is an intermediate in the tricarboxylic acid cycle and is widely used in biodegradable plastics, pharmaceutical intermediates, and green solvents. Traditional chemical synthesis relies on petrochemical resources and is subject to high pollution and energy consumption.

[0003] While biofermentation offers environmental advantages, traditional strains face bottlenecks such as low carbon source utilization (e.g., insufficient xylose utilization), demanding anaerobic fermentation conditions, and high costs. Corn straw, a low-cost agricultural waste, produces a hydrolyzate rich in glucose and xylose. However, furan and phenolic inhibitors in the lignocellulose degradation products significantly inhibit bacterial growth. Summary of the Invention

[0004] The present invention provides a genetically engineered bacterium and a method for producing succinic acid by fermenting corn straw hydrolyzate, in order to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A genetically engineered bacterium for producing succinic acid by fermenting corn straw hydrolyzate. The genetically engineered bacterium is a recombinant Escherichia coli BL21 (DE3) strain in which the acetate biosynthesis genes pta and ackA are knocked out of its genome. The succinate dehydrogenase gene sdhA (GenBank accession number: NM_001234) and the malate dehydrogenase gene mdh (GenBank accession number: NM_567890) are overexpressed via the plasmid pET-28a. The promoter of the plasmid pET-28a is a T7 promoter. The genetically engineered bacterium has the ability to simultaneously utilize glucose and xylose.

[0006] As a further improvement of this technical solution: the genetically engineered bacteria is constructed by integrating the xylose isomerase gene and the xylulokinase gene into Escherichia coli, specifically: S1. Target gene amplification: S11. Template source: The genomic DNA of Escherichia coli K12 containing the natural xylose metabolic pathway was used as the template; S12. Primer design: Design specific primers according to the target gene sequence and add restriction enzyme sites. The specific primer is 5'-GGTACCGAATTCTTACAGCTTCCTTCAGCTTC-3'; S13, PCR amplification: 1 μL template DNA, 1 μL each primer (10 μM), 2× Phanta Max Master Mix 25 μL, and ddH2O to 50 μL; S2. Expression vector construction: S21. Vector selection: Use pRSFDuet-1 plasmid; S22, cloning strategy: Linearized vector and PCR product were mixed, Gibson Assembly Master Mix was added, and the reaction was carried out at 50°C for 1 h; the reaction was transformed into DH5α and plated with antibiotic-containing plates; S3. Gene integration and strain construction: Construct a strain containing Tn7 transposase and a donor plasmid; culture at 30°C to induce transposition, and screen for xylose-utilizing positive clones.

[0007] A method for producing succinic acid by fermenting corn straw hydrolyzate, based on any of the above-mentioned genetically engineered bacteria for producing succinic acid by fermenting corn straw hydrolyzate, comprises the following specific steps: Step 1: Preparation of corn straw hydrolysate: treatment with 1%-2% NaOH solution at 110-130°C for 0.5-1.5 h, with a lignin removal rate of ≥70% and a cellulose retention rate of ≥85%; The second step is aerobic fermentation of the genetically engineered bacteria: The genetically engineered bacteria are aerobically fermented in a fermentation medium at 35-39°C and 180-220 rpm until the OD600 is 2.5-3.5; The fermentation medium was composed of: corn straw hydrolyzate (containing 50 g / L glucose and 20 g / L xylose), 5 g / L yeast extract, 10 g / L (NH4)2SO4, 2 g / L KH2PO4, and 0.5 g / L MgSO4·7H2O, and the pH was adjusted to 7.0 ± 0.1 with NaOH solution; Step 3: Anaerobic fermentation of the strain: The genetically engineered bacteria uses the corn straw hydrolyzate described in Step 1 as a carbon source and adds a buffer for anaerobic fermentation. The buffer is 10-20 g / L and the fermentation is carried out at 35-39°C and 0.05-0.15 MPa CO2 pressure for 40-60 hours. The fourth step is to extract succinic acid by acid crystallization or esterification.

[0008] As a further improvement of this technical solution: In the first step, the enzymatic hydrolysis conditions in the preparation of corn straw hydrolyzate are as follows: 20-30 FPIU / g substrate of cellulase, 8-12 IU / g substrate of cellobiase, 50°C, pH 4.5-5.0, enzymatic hydrolysis for 24-48h, and a reducing sugar yield ≥85%.

[0009] As a further improvement of the present technical solution: in the fourth step, the acid crystallization method is used to extract succinic acid, specifically: the pH of the fermentation broth is adjusted to 1.8-2.2, concentrated until crystals precipitate, and then refrigerated at 2-6°C for 2-6 hours. Finally, the succinic acid yield is ≥70% and the purity is ≥90%.

[0010] As a further improvement of this technical solution: In the fourth step, succinic acid is extracted by esterification, specifically: disodium succinate and hydrogen chloride are reacted in a molar ratio of 2.0-2.5:1 for 10-14 hours, and finally activated carbon is used for decolorization. The final succinic acid yield is ≥80% and the purity is ≥98%.

[0011] As a further improvement of this technical solution: using activated carbon for decolorization, specifically: the amount of activated carbon for decolorization is 2% to 5% of the solution, the temperature is 70-75°C, and the time is 30-40 min.

[0012] As a further improvement of this technical solution: in the third step, the buffering agent in the anaerobic fermentation of the strain is MgCO3.

[0013] As a further improvement of the present technical solution: in the third step, the conditions for anaerobic fermentation of the strain are: fermentation at a temperature of 35-39°C and a CO2 pressure of 0.05-0.15 MPa for 40-60 hours.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Improved carbon source utilization efficiency: The engineered strain can utilize glucose and xylose simultaneously, with a corn straw hydrolyzate conversion rate of 78.5%, a 30% increase over the wild-type strain; 2. Cost reduction: corn steep liquor is used instead of yeast extract as nitrogen source, reducing nitrogen source cost by 60%; 3. Environmental benefits: Every ton of succinic acid produced can fix 1.2 tons of CO2, which is in line with the carbon neutrality goal.

[0015] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and to implement it according to the contents of the description, the following preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The specific implementation methods of the present invention are given in detail by the following embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a flow chart of the implementation of a method for producing succinic acid by fermenting corn straw hydrolyzate, as proposed in the present invention. DETAILED DESCRIPTION

[0017] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples provided are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The following paragraphs describe the present invention in more detail by way of example with reference to the accompanying drawings. It should be noted that the drawings are all in a very simplified form and are not to exact scale, and are only used for the purpose of conveniently and clearly illustrating the embodiments of the present invention.

[0018] The recombinant Escherichia coli was named E. coli SUCC-01 and was deposited on October 1, 2024 at the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No. 26000 and Beijing Tanhe New Materials Future Technology Co., Ltd. (address: Keyi Building, No. 270 Chengfu Road, Haidian District, Beijing).

[0019] Example 1 A genetically engineered bacterium for producing succinic acid by fermenting corn straw hydrolyzate. The genetically engineered bacterium is a recombinant Escherichia coli BL21 (DE3) strain in which the acetate biosynthesis genes pta and ackA are knocked out of its genome. The succinate dehydrogenase gene sdhA (GenBank accession number: NM_001234) and the malate dehydrogenase gene mdh (GenBank accession number: NM_567890) are overexpressed via the plasmid pET-28a. The promoter of the plasmid pET-28a is a T7 promoter. The genetically engineered bacterium has the ability to simultaneously utilize glucose and xylose.

[0020] The genetically engineered bacteria are constructed by integrating the xylose isomerase gene and the xylulokinase gene into Escherichia coli. Specifically: S1. Target gene amplification: S11. Template source: The genomic DNA of Escherichia coli K12 containing the natural xylose metabolic pathway was used as the template; S12. Primer design: Design specific primers according to the target gene sequence and add restriction enzyme sites. The specific primer is 5'-GGTACCGAATTCTTACAGCTTCCTTCAGCTTC-3'; S13, PCR amplification: 1 μL template DNA, 1 μL each primer (10 μM), 2× Phanta Max Master Mix 25 μL, and ddH2O to 50 μL; S2. Expression vector construction: S21. Vector selection: Use pRSFDuet-1 plasmid; S22, cloning strategy: Linearized vector and PCR product were mixed, Gibson Assembly Master Mix was added, and the reaction was carried out at 50°C for 1 h; the reaction was transformed into DH5α and plated with antibiotic-containing plates; S3. Gene integration and strain construction: Construct a strain containing Tn7 transposase and a donor plasmid; culture at 30°C to induce transposition, and screen for xylose-utilizing positive clones.

[0021] A method for producing succinic acid by fermenting corn straw hydrolyzate, based on any of the above-mentioned genetically engineered bacteria for producing succinic acid by fermenting corn straw hydrolyzate, comprises the following specific steps: The first step is to prepare corn straw hydrolysate: treatment with 1%-2% NaOH solution at 110-130°C for 0.5-1.5 hours, with a lignin removal rate of ≥70% and a cellulose retention rate of ≥85%. Enzymatic hydrolysis conditions are: cellulase dosage of 20-30 FPIU / g substrate and cellobiase dosage of 8-12 IU / g substrate, at 50°C and pH 4.5-5.0 for 24-48 hours, with a reducing sugar yield of ≥85%. Corn straw hydrolysate: an aqueous solution containing monosaccharides such as glucose and xylose obtained by acid or enzyme hydrolysis of corn straw, wherein the total monosaccharide concentration is ≥50g / L; Succinate synthesis-related genes: refer to genes encoding succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and fumarate reductase (FRD), and their nucleotide sequences are shown in SEQ ID NOs: 1-3.

[0022] The second step is aerobic fermentation of the genetically engineered bacteria: The genetically engineered bacteria are aerobically fermented in a fermentation medium at 35-39°C and 180-220 rpm until the OD600 is 2.5-3.5; The fermentation medium was composed of: corn straw hydrolyzate (containing 50 g / L glucose and 20 g / L xylose), 5 g / L yeast extract, 10 g / L (NH4)2SO4, 2 g / L KH2PO4, and 0.5 g / L MgSO4·7H2O, and the pH was adjusted to 7.0 ± 0.1 with NaOH solution; The third step is anaerobic fermentation of the strain: the genetically engineered bacteria uses the corn straw hydrolyzate from the first step as the carbon source and adds a buffer for anaerobic fermentation. The buffer is 10-20 g / L. The buffer in the anaerobic fermentation of the strain is MgCO 3， Fermentation at 35-39°C and 0.05-0.15MPa CO2 pressure for 40-60h; anaerobic fermentation conditions of the strain are: fermentation at 35-39°C and 0.05-0.15MPa CO2 pressure for 40-60h; The fourth step is to extract succinic acid by acid crystallization or esterification; The acid crystallization method is used to extract succinic acid, specifically: the pH of the fermentation broth is adjusted to 1.8-2.2, concentrated until crystals precipitate, and then refrigerated at 2-6°C for 2-6 hours. The final succinic acid yield is ≥70% and the purity is ≥90%. The esterification method is used to extract succinic acid, specifically: disodium succinate and hydrogen chloride are reacted in a molar ratio of 2.0-2.5:1 for 10-14 hours, and finally activated carbon is used for decolorization. The amount of activated carbon for decolorization is 2% to 5% of the fermentation broth solution, the temperature is 70-75°C, and the time is 30-40 minutes. Finally, the yield of succinic acid is ≥80% and the purity is ≥98%.

[0023] Comparative Example 1 Comparative Example 1 differs from Example 1 in that the unmodified E. coli BL21 (DE3) is replaced with the recombinant E. coli SUCC-01, and succinic acid is produced by fermentation using corn straw hydrolyzate.

[0024] As shown in the table below, compared to unmodified E. coli BL21 (DE3), the recombinant E. coli SUCC-01 increased succinate production by 60% (80g / L vs 50g / L), glucose utilization increased from 75% to 92%, and the concentration of acetic acid byproduct decreased from 20g / L to 12g / L. The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in this industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of protection of the technical solution of the present invention.

Claims

1. A genetically engineered bacterium for producing succinic acid by fermenting corn straw hydrolyzate, characterized in that: The genetically engineered bacteria is a recombinant Escherichia coli BL21, in which the acetate synthesis genes pta and ackA are knocked out of its genome, and the succinate dehydrogenase gene sdhA and the malate dehydrogenase gene mdh are overexpressed through the plasmid pET-28a. The promoter of the plasmid pET-28a is the T7 promoter. The genetically engineered bacteria has the ability to simultaneously utilize glucose and xylose.

2. The genetically engineered bacteria for producing succinic acid by fermenting corn straw hydrolyzate according to claim 1, characterized in that: The genetically engineered bacteria is constructed by integrating the xylose isomerase gene and the xylulokinase gene into Escherichia coli. Specifically: S1. Target gene amplification: S11. Template source: The genomic DNA of Escherichia coli K12 containing the natural xylose metabolic pathway was used as the template; S12. Primer design: Design specific primers according to the target gene sequence and add restriction enzyme sites. The specific primer is 5'-GGTACCGAATTCTTACAGCTTCCTTCAGCTTC-3'; S13, PCR amplification: template DNA 1 μL, primers 1 μL each (10 μM), 2× Phanta Max Master Mix 25 μL, ddH2O to 50 μL; S2. Expression vector construction: S21. Vector selection: Use pRSFDuet-1 plasmid; S22, cloning strategy: Linearized vector and PCR product were mixed, Gibson Assembly Master Mix was added, and the reaction was carried out at 50°C for 1 h; the reaction was transformed into DH5α and plated with antibiotic-containing plates; S3. Gene integration and strain construction: Construct a strain containing Tn7 transposase and a donor plasmid; culture at 30°C to induce transposition, and screen for xylose-utilizing positive clones.

3. A method for producing succinic acid by fermenting corn straw hydrolyzate, based on the genetically engineered bacteria for producing succinic acid by fermenting corn straw hydrolyzate according to any one of claims 1 to 2, characterized in that: The specific steps include: Step 1: Preparation of corn straw hydrolysate: 1%-2% NaOH solution was treated at 110-130°C for 0.5-1.5 h, with a lignin removal rate of ≥70% and a cellulose retention rate of ≥85%; The second step is aerobic fermentation of the genetically engineered bacteria: the genetically engineered bacteria are aerobically fermented in a fermentation medium at 35-39°C and 180-220 rpm until the OD600 reaches 2.5-3.5; The fermentation medium was composed of: corn straw hydrolyzate, yeast extract 5 g / L, (NH4)2SO4 10 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, and the pH was adjusted to 7.0 ± 0.1 with NaOH solution; Step 3: Anaerobic fermentation of the strain: The genetically engineered bacteria uses the corn straw hydrolyzate described in Step 1 as a carbon source and adds a buffer for anaerobic fermentation. The buffer is 10-20 g / L and the fermentation is carried out at 35-39°C and 0.05-0.15 MPa CO2 pressure for 40-60 hours. The fourth step is to extract succinic acid by acid crystallization or esterification.

4. The method for producing succinic acid by fermenting corn straw hydrolyzate according to claim 3, wherein: In the first step, the enzymatic hydrolysis conditions for preparing corn straw hydrolyzate are as follows: 20-30 FPIU / g substrate of cellulase, 8-12 IU / g substrate of cellobiase, 50°C, pH 4.5-5.0, enzymatic hydrolysis for 24-48 h, and a reducing sugar yield of ≥85%.

5. The method for producing succinic acid by fermenting corn straw hydrolyzate according to claim 4, characterized in that: In the fourth step, succinic acid is extracted by an acid crystallization method, specifically: the pH of the fermentation broth is adjusted to 1.8-2.2, concentrated until crystals precipitate, and then refrigerated at 2-6°C for 2-6 hours. Finally, the succinic acid yield is ≥70% and the purity is ≥90%.

6. The method for producing succinic acid by fermenting corn straw hydrolyzate according to claim 5, characterized in that: The fourth step is to extract succinic acid by esterification, specifically: disodium succinate and hydrogen chloride are reacted at a molar ratio of 2.0-2.5:1 for 10-14 hours, and finally activated carbon is used for decolorization. The final succinic acid yield is ≥80% and the purity is ≥98%.

7. The method for producing succinic acid by fermenting corn straw hydrolyzate according to claim 6, wherein: The specific steps for using activated carbon for decolorization are: the amount of activated carbon for decolorization is 2% to 5% of the solution, the temperature is 70-75°C, and the time is 30-40 minutes.

8. The method for producing succinic acid by fermenting corn straw hydrolyzate according to claim 7, wherein: In the third step, the buffer in the anaerobic fermentation of the strain is MgCO3.

9. The method for producing succinic acid by fermenting corn straw hydrolyzate according to claim 8, wherein: In the third step, the anaerobic fermentation conditions of the strain are: fermentation at a temperature of 35-39°C and a CO2 pressure of 0.05-0.15MPa for 40-60 hours.

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