EP-4737557-A1 - MODIFIED ESCHERICHIA COLI AND USE THEREOF IN PRODUCTION OF L-AMINO ACID BY MEANS OF FERMENTATION

EP4737557A1EP 4737557 A1EP4737557 A1EP 4737557A1EP-4737557-A1

Abstract

A modified Escherichia coli and the use thereof in the production of L-amino acid by means of fermentation. Specifically, disclosed is the use of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase and malate dehydrogenase in the construction of a genetically engineered bacterium for producing L-amino acid. Experiments prove that the recombinant bacterium, which is conducive to the accumulation of L-amino acid, is obtained by means of knocking out the encoding genes of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and the malate dehydrogenase of Escherichia coli capable of producing L-amino acid, and introducing the encoding gene of the branched-chain amino acid transaminase derived from Bacillus subtilis. The recombinant bacteria can greatly increase the yield of L-amino acid and reduce the costs, and is of great significance to accelerate the industrial progress of L-amino acid.

Inventors

MENG, GANG
WEI, Aiying
ZHAO, Chunguang
SU, Houbo
ZHANG, YING
BI, Guodong
ZHANG, XIAOQIN
WANG, Pan
FU, LIXIA

Assignees

Ningxia Eppen Biotech Co. Ltd

Dates

Publication Date: 20260506
Application Date: 20240624

Claims (13)

Use of a protein combination, which is at least one of A1)-A5): A1) the construction of genetically engineered bacteria that produce L-amino acid; A2) the production of L-amino acid; A3) the regulation of the yield of L-amino acid; A4) the preparation of a product for producing L-amino acid; and A5) the preparation of food, feed or drug containing L-amino acid, the protein combination comprises at least one of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase and malate dehydrogenase; the pyruvate formate lyase is B1) or B2) or B3): B1) a protein comprising an amino acid sequence as shown in SEQ ID No. 13; B2) a protein which is obtained by substitution and/or deletion and/or addition of amino acid residue(s) in the amino acid sequence as shown in SEQ ID No. 13, has 90% or more identity with the protein shown in B1) and has the same function as the protein shown in B1); B3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of B1) or B2) and has the same function as that of B1) or B2); the alcohol dehydrogenase is C1) or C2) or C3): C1) a protein comprising an amino acid sequence as shown in SEQ ID No. 14; C2) a protein which is obtained by substitution and/or deletion and/or addition of amino acid residue(s) in the amino acid sequence as shown in SEQ ID No. 14, has 90% or more identity with the protein shown in C1) and has the same function as the protein shown in C1); C3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of C1) or C2) and has the same function as that of C1) or C2); the branched-chain amino acid transaminase derived from Escherichia coli is D1) or D2) or D3): D1) a protein comprising an amino acid sequence as shown in SEQ ID No. 15; D2) a protein which is obtained by substitution and/or deletion and/or addition of amino acid residue(s) in the amino acid sequence as shown in SEQ ID No. 15, has 90% or more identity with the protein shown in D1) and has the same function as the protein shown in D1); D3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of D1) or D2) and has the same function as that of D1) or D2); the branched-chain amino acid transaminase derived from Bacillus subtilis is E1) or E2) or E3): E1) a protein comprising an amino acid sequence as shown in SEQ ID No. 16; E2) a protein which is obtained by substitution and/or deletion and/or addition of amino acid residue(s) in the amino acid sequence as shown in SEQ ID No. 16, has 90% or more identity with the protein shown in E1) and has the same function as the protein shown in E1); E3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of E1) or E2) and has the same function as that of E1) or E2); the thiamine phosphate synthase is F1) or F2) or F3): F1) a protein comprising an amino acid sequence as shown in SEQ ID No. 17; F2) a protein which is obtained by substitution and/or deletion and/or addition of amino acid residue(s) in the amino acid sequence as shown in SEQ ID No. 17, has 90% or more identity with the protein shown in F1) and has the same function as the protein shown in F1); F3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of F1) or F2) and has the same function as that of F1) or F2); the malate dehydrogenase is G1) or G2) or G3): G1) a protein comprising an amino acid sequence as shown in SEQ ID No. 18; G2) a protein which is obtained by substitution and/or deletion and/or addition of amino acid residue(s) in the amino acid sequence as shown in SEQ ID No. 18, has 90% or more identity with the protein shown in G1) and has the same function as the protein shown in G1); and G3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of G1) or G2) and has the same function as that of G1) or G2).
Use of a nucleic acid molecule that encodes the protein combination of claim 1, which is at least one A1)-A5): A1) the construction of genetically engineered bacteria that produce L-amino acid; A2) the production of L-amino acid; A3) the regulation of the yield of L-amino acid; A4) the preparation of a product for producing L-amino acid; and A5) the preparation of food, feed or drug containing L-amino acid.
Use of a substance that inhibits or down-regulates the expression amount and/or activity of the pyruvate formate lyase according to claim 1; and/or a substance that inhibits or down-regulates the expression amount and/or activity of the alcohol dehydrogenase according to claim 1; and/or a substance that inhibits or down-regulates the expression amount and/or activity of the branched-chain amino acid transaminase derived from Escherichia coli according to claim 1; and/or a substance that inhibits or down-regulates the expression amount and/or activity of the thiamine phosphate synthase according to claim 1; and/or or a substance that inhibits or down-regulates the expression amount and/or activity of the malate dehydrogenase according to claim 1; and/or a substance that increases or up-regulates the expression amount and/or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis according to claim 1, which is at least one of A1)-A5): A1) the construction of genetically engineered bacteria that produce L-amino acid; A2) the production of L-amino acid; A3) the regulation of the yield of L-amino acid; A4) the preparation of a product for producing L-amino acid; and A5) the preparation of food, feed or drug containing L-amino acid.
Use of an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant host cell, which contains the nucleic acid molecule according to claim 2, the substance that inhibits or down-regulates the expression amount and/or activity of the pyruvate formate lyase according to claim 1, the substance that inhibits or down-regulates the expression amount and/or activity of the alcohol dehydrogenase according to claim 1, the substance that inhibits or down-regulates the expression amount and/or activity of the branched-chain amino acid transaminase derived from Escherichia coli according to claim 1, the substance that inhibits or down-regulates the expression amount and/or activity of the thiamine phosphate synthase according to claim 1, the substance that inhibits or down-regulates the expression amount and/or activity of the malate dehydrogenase according to claim 1, and/or the substance that increases or up-regulates the expression amount and/or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis according to claim 1, which is at least one of A1)-A5): A1) the construction of genetically engineered bacteria that produces L-amino acid; A2) the production of L-amino acid; A3) the regulation of the yield of L-amino acid; A4) the preparation of a product for producing L-amino acid; and A5) the preparation of food, feed or drug containing L-amino acid.
A recombinant bacterium, characterized in that , the recombinant bacterium is obtained by weak expression or no expression of at least one of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and the malate dehydrogenase of claim 1 in originating bacteria; and/or expression or over-expression of the branched-chain amino acid transaminase derived from Bacillus subtilis of claim 1.
The recombinant bacterium according to claim 5, characterized in that , the weak expression or no expression is implemented by reducing the expression amount and/or activity of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and/or the malate dehydrogenase in the originating bacteria; and the expression or over-expression is implemented by introducing a coding gene of the branched-chain amino acid transaminase derived from Bacillus subtilis into the originating bacteria.
The recombinant bacterium according to claim 6, characterized in that , the reducing the expression amount and/or activity of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and/or the malate dehydrogenase in the originating bacteria are implemented by a gene editing, gene knockout, gene mutation or gene attenuation technology, causing a decrease in the expression amount, activity reduction or inactivation of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and/or the malate dehydrogenase in the originating bacteria.
A method for increasing the yield of L-amino acid, comprising the following steps: reducing the expression amount and/or activity of at least one of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and the malate dehydrogenase of claim 1 in originating bacteria; and/or increasing the expression amount and/or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis of claim 1 to obtain a recombinant bacterium, the yield of L-amino acid in the recombinant bacterium is higher than that of the originating bacteria.
The method according to claim 8, characterized in that , the reducing the expression amount and/or activity of at least one of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and the malate dehydrogenase of claim 1 in the originating bacteria is achieved by a gene editing, gene knockout, gene mutation or gene attenuation technology, causing a decrease in the expression amount, activity reduction or inactivation of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and/or the malate dehydrogenase in the originating bacteria. the increasing the expression amount and/or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis of claim 1 is implemented by introducing a coding gene of the branched-chain amino acid transaminase derived from Bacillus subtilis into the originating bacteria.
A method for producing L-amino acid, comprising the following steps: fermenting and culturing the recombinant bacterium of any one of claims 5 to 7, and collecting a fermentation product to obtain L-amino acid therefrom.
The use according to any one of claims 1 to 4 or the method according to claim 8 or 10, characterized in that , the L-amino acid is L-valine.
The recombinant bacterium according to any one of claims 5 to 7 or the method according to claim 8 or 9, characterized in that , the originating bacterium is Escherichia coli.
Use of the recombinant bacterium of any one of claims 5 to 7, which is at least one of A2)-A5): A2) the production of L-amino acid; A3) the regulation of the yield of L-amino acid; A4) the preparation of a product for producing L-amino acid; and A5) the preparation of food, feed or drug containing L-amino acid.

Description

Cross-reference to Related Application This application claims priority to the following four patent applications: Chinese patent application 202310765899.5 filed to the China Patent Office on June 27, 2023 and titled "Recombinant Escherichia Coli for fermentation and production of L-valine"; Chinese patent application 202310784984.6 filed to the China Patent Office on June 29, 2023 and titled "Modified Escherichia coli and use thereof in production of L-valine by means of fermentation"; Chinese patent application 202310784863.1 filed to the Chinese Patent Office on June 29, 2023 and titled "Recombinant bacterium and use thereof in production of L-valine"; and Chinese patent application 202310809284.8 filed to the Chinese Patent Office on July 04, 2023 and titled "Method for Increasing Yield of L-valine and Recombinant Bacterium Used in Method", all of which are incorporated herein by reference in their entireties. Technical Field The present invention belongs to the field of biotechnology, and relates to modified Escherichia coli and use thereof in the production of L-amino acid by means of fermentation. Background Art L-valine has important applications in food, medicine, health products, feed and other fields. In the field of food, L-valine can supplement nutrients, promote body growth, and provide energy. In the field of medicine, L-valine may be used in infusions and injections to promote surgical wound healing. The export volume of feed-grade L-valine in China reaches more than 30,000 tons, with an average annual growth rate of 20%. At present, many domestic enterprises have carried out the production of L-valine through a fermentation method, and many laboratories are also conducting research projects on the fermentation process of L-valine. The researchers have found that the process of intracellular L-valine secretion through a cell membrane to the outside of a cell requires the participation of specific transporters, but in this process, the activity of specific transporters cannot meet the timely secretion of intracellular valine through the cell membrane to the outside of the cell, resulting in accumulation of intracellular L-valine; at the same time, acetohydroxyacid synthase is contained in bacterial cells, has a catalytic effect on the biosynthesis of L-valine and can promote acid production, but when L-valine synthesized in the bacterial cells cannot be excreted and accumulated in time, acetohydroxyacid synthase is strongly inhibited by L-valine to reduce its catalytic effect, and the synthesis of L-valine cannot be further catalyzed, so the continued synthesis of L-valine is limited, which ultimately leads to low yield. In addition, studies have found that Escherichia coli is very sensitive to L-valine, and when L-valine in L-valine producing bacteria cannot be excreted in time and accumulates, the growth of Escherichia coli cannot be seriously restricted, which in turn leads to the inhibition of L-valine synthesis in bacterial cells, resulting in low yield. Summary of the Invention The main purpose of the present invention is how to increase the yield of L-amino acid. The purpose of the present invention is not limited to the described subject. Other purposes which are not mentioned herein can be clearly understood by those skilled in the art through the following description. The present invention protects use of a protein combination first, which may be at least one of A1)-A5): A1) the construction of genetically engineered bacteria that produces L-amino acid;A2) the production of L-amino acid;A3) the regulation of the yield of L-amino acid;A4) the preparation of a product for producing L-amino acid; andA5) the preparation of food, feed or drug containing L-amino acid. In the above-mentioned use, the protein combination may include at least one of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase and malate dehydrogenase. In the above-mentioned use, the protein combination may specifically be composed of at least one of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase and malate dehydrogenase. Any of the above-mentioned pyruvate formate lyase is B1) or B2) or B3): B1) a protein having an amino acid sequence as shown in SEQ ID No. 13;B2) a protein which is obtained by substitution and/or deletion and/or addition of an amino acid residue in the amino acid sequence as shown in SEQ ID No. 13, has the identity of 90% or more with the protein shown in B1) and has the same function as the protein shown in B1); andB3) a fusion protein which is obtained by linking a tag to an N-terminal or/and a C-terminal of B1) or B2) and has the same function as that of B1) or B2). A