CN108728470B

CN108728470B - Recombinant bacterium for producing beta-alanine and construction method and application thereof

Info

Publication number: CN108728470B
Application number: CN201710247567.2A
Authority: CN
Inventors: 刘伟丰; 刘姣; 刘波; 薛燕芬; 陶勇
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2017-04-14
Filing date: 2017-04-14
Publication date: 2022-01-11
Anticipated expiration: 2037-04-14
Also published as: CN108728470A

Abstract

The invention discloses a recombinant bacterium for producing beta-alanine and a construction method and application thereof. The construction method of the recombinant bacteria disclosed by the invention comprises the following steps: knocking out fadR gene, fabF gene, fabH gene, iclR gene and sucA gene of recipient bacterium, introducing aspC gene or gene cluster, panD gene, alkL gene and gdh gene into the recipient bacterium, and enhancing the expression of fadL gene, fadD gene, sthA gene, atoSC gene cluster and aceBA gene cluster in the recipient bacterium; the recipient bacterium is a bacterium or fungus containing a fadR gene, a fabF gene, a fabH gene, an iclR gene and a sucA gene. Experiments prove that the conversion rate of beta-alanine produced by using the recombinant bacterium as a raw material and using fatty acid as a raw material is 60.87%, which indicates that the recombinant bacterium can be used for preparing beta-alanine.

Description

Recombinant bacterium for producing beta-alanine and construction method and application thereof

Technical Field

The invention relates to a recombinant bacterium for producing beta-alanine and a construction method and application thereof in the field of biotechnology.

Background

beta-Alanine (the english name is beta-Alanine), also known as beta-aminopropionic acid, is a non-protein amino acid with important value. The beta-alanine is used as a biochemical raw material and has wide application prospect in the fields of medicines, feeds, foods and the like. For example, beta-alanine can be used to synthesize pantothenic acid (vitamin B5), an integral part of coenzyme A which is essential for various metabolisms.

At present, the synthesis method of beta-alanine mainly comprises a chemical synthesis method and a biological conversion method, and specifically comprises the following steps:

1. chemical synthesis method

(1) An acrylic acid method: the beta-alanine is obtained mainly by carrying out amination reaction on acrylic acid (or acrylate) and ammonia water at high temperature and pressure. The main problem of the acrylic acid method is that many by-products are produced and high temperature and high pressure are required. The acrylic acid has strong corrosivity and higher requirements on equipment.

(2) The acrylonitrile method: including direct ammoniation and ammoniation hydrolysis. The direct ammoniation method adopts the one-step reaction of alkene nitrile and ammonia water at high temperature and high pressure to synthesize beta-alanine; the ammonification hydrolysis method is that acrylonitrile reacts with ammonia at high temperature and high pressure to generate aminopropionitrile, and then the acrylonitrile is hydrolyzed under acidic or alkaline conditions to generate beta-alanine. The method also has higher requirements on equipment due to the requirement of high temperature and high pressure, and simultaneously needs higher safety protection measures due to the fact that the used acrylonitrile is a highly toxic raw material. The method has low yield, and the product purity is not high due to the generation of a large amount of inorganic salt in the hydrolysis process.

(3) Beta-aminopropionitrile method: beta-aminopropionitrile hydrolyzes under acidic or basic conditions to generate beta-alanine. The aminopropionitrile method has the characteristics of high reaction yield and the defects of higher price of beta-aminopropionitrile and generation of a large amount of inorganic salt in the hydrolysis process.

2. Biotransformation process

The biotransformation method is mainly to obtain beta-alanine by enzymatic conversion or whole cell catalysis by expressing relevant enzymes. The catalytic action of the following two enzymes is mainly adopted to convert different substrates into beta-alanine.

(1) The acrylic acid ammonifying enzyme method comprises the following steps: acrylic acid is converted into beta-alanine by using beta-acrylic acid ammonifying enzyme expressed by sarcina lutea, but the raw material acrylic acid is strong corrosive and irritant liquid, has higher requirements on personnel safety and equipment, and has no report of industrial application at present.

(2) L-aspartic acid-alpha-decarboxylase: the cost of the process, which converts L-aspartic acid to-alanine using L-aspartate-alpha-decarboxylase, depends on the cost of the L-aspartic acid starting material.

In summary, the chemical synthesis method for producing beta-alanine generally faces the problems of harsh reaction conditions, difficulty in separation and purification, easy environmental pollution and the like. The production of beta-alanine by biotransformation requires the establishment of a cheap raw material route to reduce the production cost and form a production mode with popularization prospect. Fatty acids are a class of substances with a highly reduced state, and fatty acid raw materials for bioconversion can be obtained at a low price from oil crude products, illegal cooking oil, and the like.

Disclosure of Invention

The technical problem to be solved by the invention is how to produce beta-alanine.

In order to solve the technical problems, the invention firstly provides a construction method of the recombinant bacteria.

The construction method of the recombinant bacterium provided by the invention comprises the steps of carrying out A or B transformation on a receptor bacterium to obtain the recombinant bacterium; the nails are A1 and A8; the B is all or part of the six types of A1, A8 and A2-A7;

a1, knocking out fatty acid degradation transcription factor fadR gene of the receptor bacterium, or inhibiting the expression of the fadR gene or inhibiting the activity of protein encoded by the fadR gene;

a2, knocking out the fabF gene of the beta-ketoacyl-ACP synthase II gene of the recipient bacterium, or inhibiting the expression of the fabF gene or inhibiting the activity of a protein encoded by the fabF gene;

a3, knocking out the fabH gene of the beta-ketoacyl-ACP synthase III gene of the recipient bacterium, or inhibiting the expression of the fabH gene or inhibiting the activity of a protein encoded by the fabH gene;

a4, knocking out the iclR gene of the recipient bacterium or inhibiting the expression of the iclR gene or inhibiting the activity of a protein encoded by the iclR gene;

a5, knocking out the sucA gene of the recipient bacterium or inhibiting the expression of the sucA gene or inhibiting the activity of a protein encoded by the sucA gene;

a6, increasing the content of the protein coded by the foreign alkane uptake outer membrane protein gene alkL gene in the recipient bacterium or enhancing the activity of the protein coded by the alkL gene;

a7, increasing the content of protein encoded by gene in L-aspartic acid synthesis pathway in the recipient bacterium or enhancing the activity of protein encoded by gene in the L-aspartic acid synthesis pathway;

a8, increasing the content of or enhancing the activity of a protein encoded by an L-aspartate- α -decarboxylase panD gene in the recipient bacterium;

the recipient bacterium is a bacterium or fungus containing the fadR gene, the fabF gene, the fabH gene, the iclR gene and the sucA gene.

In the above method, the recipient bacterium may be 1) or 2):

1) e.coli;

2) coli BW 25113.

In the above method, the alkL gene may be derived from Haemophilus hydrocarbonoclosticus (Marinobacter hydrocarbonoclosticus) or/and Pseudomonas putida (Pseudomonas putida).

The gene in the L-aspartic acid synthesis pathway can be a gene aspC gene which is derived from Escherichia coli (Escherichia coli) or/and Alcaligenes eutrophus (Ralstonia eutropha) or/and Pseudomonas aeruginosa (Pseudomonas aeruginosa) and codes aspartate aminotransferase, a gene aspDH gene which codes L-aspartate dehydrogenase or/and a gene aspA gene which codes aspartate lyase.

The panD gene can be derived from Tribolium castaneum or/and Escherichia coli or/and Bacillus subtilis.

In the above method, the fadR gene may encode the following proteins a1) or a 2):

a1) protein shown as SEQ ID No. 2in a sequence table;

a2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No. 2in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.2 and has the same function.

The fabF gene may encode the following proteins a3) or a 4):

a3) protein shown as SEQ ID No.14 in a sequence table;

a4) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.14 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.14 and has the same function.

The fabH gene can encode the following proteins a5) or a 6):

a5) protein shown as SEQ ID No.16 in the sequence table;

a6) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.16 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.16 and has the same function.

The iclR gene can code the following proteins a7) or a 8):

a7) protein shown as SEQ ID No.39 in a sequence table;

a8) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.39 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.39 and has the same function.

The sucA gene may encode the following proteins a9) or a 10):

a9) protein shown by SEQ ID No.41 in a sequence table;

a10) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.41 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.41 and has the same function.

The alkL gene can encode the following proteins a11) or a 12):

a11) protein shown as SEQ ID No.29 in the sequence table;

a12) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.29 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.29 and has the same function.

The aspC gene may encode the following proteins a13) or a 14):

a13) protein shown as SEQ ID No.48 in the sequence table;

a14) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.48 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.48 and has the same function.

The panD gene can encode the following proteins a15) or a 16):

a15) protein shown as SEQ ID No.52 in the sequence table;

a16) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.52 in the sequence table, has 75 percent or more than 75 percent of identity with the amino acid sequence of SEQ ID No.52 and has the same function.

In the above method, A6 can be obtained by introducing the alkL gene into the recipient bacterium.

A7 can be obtained by introducing the gene in the L-aspartic acid synthesis pathway into the recipient bacterium.

A8 can be achieved by introducing the panD gene into the recipient bacterium.

In the above method, the introduction of the alkL gene into the recipient bacterium may specifically be the introduction of an expression vector containing the alkL gene (i.e., an alkL gene expression vector) into the recipient bacterium.

The introduction of the gene in the L-aspartic acid synthesis pathway into the recipient bacterium may specifically be the introduction of an expression vector comprising the gene in the L-aspartic acid synthesis pathway (i.e., an expression vector for the gene in the L-aspartic acid synthesis pathway) into the recipient bacterium.

The introduction of the panD gene or gene cluster into the recipient bacterium may specifically be the introduction of an expression vector comprising the panD gene or gene cluster (i.e., a panD gene or gene cluster expression vector) into the recipient bacterium.

The expression vectors can be plasmid, cosmid, phage, or viral vectors. The plasmid can be pLB1a or pXB1k, the sequence of pLB1a is SEQ ID No.24 in the sequence table, and the sequence of pXB1k is SEQ ID No.35 in the sequence table.

When the alkL gene, the gene in the L-aspartic acid synthesis pathway and/or the panD gene are introduced into the recipient bacterium, they may be achieved by introducing a separate expression vector containing only one of the alkL gene, the gene in the L-aspartic acid synthesis pathway and the panD gene, or by introducing a co-expression vector containing at least two of the alkL gene, the gene in the L-aspartic acid synthesis pathway and the panD gene.

In one embodiment of the present invention, the gene in the L-aspartic acid synthesis pathway is the aspC gene, and the introduction of the alkL gene and the aspC gene into the recipient bacterium is carried out by introducing a co-expression vector containing both the genes (i.e., an alkL-aspC co-expression vector) into the recipient bacterium, and the introduction of the panD gene into the recipient bacterium is carried out by introducing a separate expression vector containing the gene (i.e., a panD co-expression vector) into the recipient bacterium. The alkL-aspC co-expression vector can be specifically a recombinant vector pLB1a-alkL-aspC obtained by introducing the alkL gene and the aspC gene into pLB1 a. The pLB1a-alkL-aspC can express aspC protein shown in SEQ ID No.48 and alkL protein shown in SEQ ID No. 29. The panD expression vector can be specifically a recombinant vector pXB1k-panD obtained by introducing the panD gene into the pXB1 k. The pXB1k-panD can express the panD protein shown in SEQ ID No. 52.

In the above method, the fadR gene may be b1) or b 2):

b1) a cDNA molecule or DNA molecule shown as SEQ ID No.1 in a sequence table;

b2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b1) and has the same function.

The fabF gene may be the following b3) or b 4):

b3) a cDNA molecule or DNA molecule shown as SEQ ID No.13 in the sequence table;

b4) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b3) and has the same function.

The fabH gene may be the following b5) or b 6):

b5) a cDNA molecule or DNA molecule shown as SEQ ID No.15 in the sequence table;

b6) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b5) and has the same function.

The iclR gene can be b7) or b8) as follows:

b7) a cDNA molecule or DNA molecule shown as SEQ ID No.38 in the sequence table;

b8) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b7) and has the same function.

The sucA gene may be the following b9) or b 10):

b9) a cDNA molecule or DNA molecule shown as SEQ ID No.40 in the sequence table;

b10) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b9) and has the same function.

The alkL gene may be b11) or b12) below:

b11) a cDNA molecule or DNA molecule shown as SEQ ID No.28 in the sequence table;

b12) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b11) and has the same function.

The aspC gene may be the following b13) or b 14):

b13) a cDNA molecule or DNA molecule shown as SEQ ID No.47 in the sequence table;

b14) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b13) and has the same function.

The panD gene can be b15) or b16) as follows:

b15) a cDNA molecule or DNA molecule shown as SEQ ID No.51 in the sequence table;

b16) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b15) and has the same function.

In the method, the knockout of the fatty acid degradation transcription factor fadR gene of the recipient bacterium described in A1 can be performed by homologous recombination, and specifically can be realized by using an Escherichia coli strain JW1176 with the fadR gene knockout character.

A2, the knockout of the fabF gene of the beta-ketoacyl-ACP synthase II gene of the recipient bacterium can be realized by homologous recombination, and specifically, the knockout of the fabF gene can be realized by an Escherichia coli strain JW1081 with the character of the fabF gene knockout.

A3, the knockout of the fabH gene of the beta-ketoacyl-ACP synthase III gene of the recipient bacterium can be realized by homologous recombination, and specifically, the knockout of the fabH gene can be realized by an escherichia coli strain JW1077 with the characteristic of the fabH gene knockout.

The knockout of the iclR gene of the recipient bacterium described in A4 can be carried out by homologous recombination, specifically by using Escherichia coli strain JW3978 having the iclR gene knockout trait.

The knockout of the sucA gene of the recipient bacterium described in A5 can be carried out by homologous recombination, specifically by using Escherichia coli strain JW0715 having the sucA gene knockout trait.

In the above method, the method further comprises six, any five, any four, any three, any two or any one of the following B1-B6:

b1, increasing the content of the protein coded by the gdh gene of the glutamate dehydrogenase gene in the recipient bacterium or enhancing the activity of the protein coded by the gdh gene;

b2, increasing the content of or enhancing the activity of a protein encoded by the fadL gene in the recipient bacterium;

b3, increasing the content of or enhancing the activity of a protein encoded by a gene in the fatty acid beta oxidation pathway in the recipient bacterium;

the gene in the fatty acid beta oxidation pathway is one or more genes selected from the following genes: a fadD gene encoding a fatty acyl-CoA synthase, a fadE gene encoding a fatty acyl-CoA dehydrogenase, a fadB gene encoding a 3-hydroxyacyl-CoA dehydrogenase, a fadA gene encoding a 3-ketoacyl-CoA thiolase, a fadI gene encoding a 3-ketoacyl-CoA thiolase, a fadJ gene encoding a 3-hydroxyacyl-CoA dehydrogenase, and a fadK gene encoding a short-chain fatty acyl-CoA synthase;

b4, increasing the content of a protein encoded by the sthA gene in the recipient bacterium or enhancing the activity of a protein encoded by the sthA gene;

b5, increasing the content of protein coded by gene in the short-chain fatty acid degradation pathway in the recipient bacterium or enhancing the activity of protein coded by gene in the short-chain fatty acid degradation pathway;

the gene in the short-chain fatty acid degradation pathway is B5a or B5B:

b5a, short chain fatty acid degradation regulation gene cluster atoSC gene cluster;

B5B, short chain fatty acid degradation gene cluster atoDAEB gene cluster;

b6, increasing the content of the protein coded by the aceBA gene cluster in the glyoxylate pathway in the recipient bacterium or enhancing the activity of the protein coded by the aceBA gene cluster in the glyoxylate pathway.

In the above method, the recipient bacterium may further comprise the fadL gene, the gene in the fatty acid β oxidation pathway, the sthA gene, the gene in the short-chain fatty acid degradation pathway, and/or an aceBA gene cluster in the glyoxylate pathway.

In the above method, the gdh gene may be derived from Escherichia coli (Escherichia coli) or/and Bacillus subtilis (Bacillus subtilis).

The gene in the short chain fatty acid degradation regulatory gene cluster atoSC gene cluster may be a gene atoC encoding an atoC transcription activator and/or a gene atoS encoding an atoS-sensitive histidine kinase.

The gene in the short chain fatty acid degradation gene cluster atoDAEB gene cluster can be a gene atoA gene for coding an acetoacetyl-CoA transferase alpha subunit, a gene atoD gene for coding an acetoacetyl-CoA transferase beta subunit, a gene atoE gene for coding an acetoacetate transporter and/or a gene atoB gene for coding an acetyl-CoA acetyltransferase.

The glyoxylate pathway aceBA gene cluster may comprise the following genes: the gene aceA coding for isocitrate lyase and the gene aceB coding for malate synthase.

In the above method, the gdh gene encodes the following protein a17) or a 18):

a17) protein shown by SEQ ID No.50 in a sequence table;

a18) and (b) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.50 in the sequence table.

The fadL gene can encode the following proteins a19) or a 20):

a19) protein shown by SEQ ID No.6 in a sequence table;

a20) and (b) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.6 in the sequence table.

The fadD gene can code the protein of the following a21) or a 22):

a21) protein shown as SEQ ID No.9 in a sequence table;

a22) and (b) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.9 in the sequence table.

The sthA gene may encode the following proteins a23) or a 24):

a23) protein shown by SEQ ID No.12 in a sequence table;

a24) and (b) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.12 in the sequence table.

The atoSC gene cluster may encode the proteins of a25) and a26) below:

a25) the following proteins of a251) or a 252):

a251) protein shown by SEQ ID No.19 in a sequence table;

a252) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.19 in the sequence table and has the same function;

a26) the following proteins of a261) or a 262):

a261) protein shown as SEQ ID No.21 in a sequence table;

a262) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.21 in the sequence table and has the same function.

The aceBA gene cluster can code the following proteins a27) and a 28):

a27) the following proteins of a271) or a 272):

a271) protein shown as SEQ ID No.44 in the sequence table;

a272) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID No.44 in the sequence table and has the same function;

a28) the following proteins of a281) or a 282):

a281) protein shown as SEQ ID No.46 in a sequence table;

a282) and (b) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID No.46 in the sequence table.

In the above method, B1 can be produced by introducing the gdh gene into the recipient bacterium.

B2 can be obtained by replacing the promoter of the fadL gene with promoter P_CPA1And (5) realizing.

B3 can be prepared by replacing the promoter of the gene in the fatty acid beta oxidation pathway with the promoter P_CPA1And (5) realizing.

B4 can be produced by replacing the promoter of the sthA gene with the promoter P_CPA1And (5) realizing.

B5 can be prepared by replacing the promoter of the gene in the short-chain fatty acid degradation pathway with the promoter P_CPA1And (5) realizing.

B6 can be obtained by replacing the promoter of the aceBA gene cluster with the promoter P_CPA1And (5) realizing.

In the above method, the promoter of the gene in the short-chain fatty acid degradation pathway may be a promoter of the short-chain fatty acid degradation regulatory gene cluster atoSC gene cluster or a promoter of the short-chain fatty acid degradation gene cluster atoDAEB gene cluster.

In the above method, the promoter P_CPA1Can be a nucleic acid molecule as shown in 1) or 2) or 3) below:

1) the coding sequence is a DNA molecule at the 1443-1622 th site of SEQ ID No.3 in the sequence table;

2) DNA molecule with 75% or more than 75% identity with the nucleotide sequence limited by 1) and the same function;

3) a DNA molecule which is hybridized with the nucleotide sequence defined in 1) under strict conditions and has the same function.

In the above method, the introducing the gdh gene into the recipient bacterium may specifically be introducing an expression vector containing the gdh gene (i.e., a gdh gene expression vector) into the recipient bacterium.

The expression vectors can be plasmid, cosmid, phage, or viral vectors. The plasmid can be pLB1a or pXB1k or a recombinant plasmid containing the alkL gene, the gene in the synthetic pathway of L-aspartic acid and/or the panD gene, the sequence of pLB1a is SEQ ID No.24 in the sequence table, and the sequence of pXB1k is SEQ ID No.35 in the sequence table.

In one embodiment of the present invention, the introduction of the gdh gene into the recipient bacterium is achieved by introducing a co-expression vector containing the gdh gene, the alkL gene, and the aspC gene (i.e., a gdh-alkL-aspC co-expression vector) into the recipient bacterium. The gdh-alkL-aspC co-expression vector may specifically be a recombinant vector pLB1a-aspC-gdhA-alkL obtained by introducing the gdh gene, the alkL gene and the aspC gene into the pLB1 a. The pLB1a-aspC-gdhA-alkL can express aspC protein shown in SEQ ID No.48, gdhA protein shown in SEQ ID No.50 and alkL protein shown in SEQ ID No. 29.

In the above method, the promoter of said fadL gene is replaced with a promoter P_CPA1Can be realized by a DNA fragment shown as SEQ ID No.4 in a sequence table.

Replacing the promoter of a gene in the fatty acid beta oxidation pathway with the promoter P_CPA1Can be realized by a DNA fragment shown as SEQ ID No.7 in a sequence table.

Replacing the promoter of the sthA gene with the promoter P_CPA1Can be realized by a DNA fragment shown as SEQ ID No.10 in a sequence table.

Replacing the promoter of the gene in the short-chain fatty acid degradation pathway with the promoter P_CPA1Can be realized by a DNA fragment shown as SEQ ID No.17 in a sequence table.

Replacing the promoter of the aceBA gene cluster with the promoter P_CPA1Can be realized by a DNA fragment shown as SEQ ID No.42 in a sequence table.

In the above methods, the 75% or greater than 75% identity may be 80%, 85%, 90%, or 95% or greater identity.

In order to solve the technical problems, the invention also provides a preparation method of the beta-alanine.

The preparation method of beta-alanine provided by the invention comprises the following steps: and (3) carrying out biotransformation by using the recombinant bacteria prepared by the preparation method of the recombinant bacteria and taking fatty acid as a substrate to prepare the beta-alanine.

In the above method for producing beta-alanine, the fatty acid may be palmitic acid, stearic acid, myristic acid, lauric acid, capric acid, caprylic acid and/or caproic acid.

The preparation method of the beta-alanine can be specifically used for preparing the beta-alanine by using the recombinant bacteria to carry out whole-cell catalysis on the fatty acid.

In order to solve the technical problem, the invention also provides any one of the following products Z1-Z4:

z1 and the recombinant bacterium prepared by the preparation method of the recombinant bacterium.

Z2, a protein or a protein set, being M1 or M2:

m1, M1a and M1b, M1a being the protein encoded by the panD gene; m1b is all or part of the protein encoded by the aspC gene, the protein encoded by the gdhA gene, and the protein encoded by the alkL gene;

m2, M1 and M2a described above, M2a is all or part of the protein encoded by the fadL gene, the protein encoded by the fadD gene, the protein encoded by the sthA gene, the protein encoded by the atoSC gene cluster and the protein encoded by the aceBA gene cluster.

Z3, a gene or a set of genes, being the following N1 or N2:

n1, N1a and N1b above, N1a being the panD gene, N1b being all or part of the aspC gene, the gdhA gene and the alkL gene;

n2, N1 and N2a as described above, N2a is all or part of the fadL gene, the fadD gene, the sthA gene, the atoSC gene cluster and the aceBA gene cluster.

Z4, kit of parts consisting of the promoter P_CPA1And said gene or set of genes.

In order to solve the technical problem, the invention also provides any one of the following applications of the product:

x1, producing beta-alanine;

x2, preparing and producing a beta-alanine product;

x3, degraded fatty acid;

and X4, and preparing a degraded fatty acid product.

The invention prepares the recombinant bacterium for producing the beta-alanine by taking the fatty acid as the raw material, and the recombinant bacterium can utilize the fatty acid raw material which is obtained from the crude oil product, the illegal cooking oil and the like at low cost to be used for producing the beta-alanine by microbial fermentation and biotransformation. Therefore, the recombinant bacterium has potential cost advantage in synthesizing beta-alanine from fatty acid raw materials. The conversion rate of beta-alanine produced by using the recombinant bacterium of the invention and taking fatty acid as a raw material is 60.87%, which shows that the recombinant bacterium of the invention can be used for preparing beta-alanine.

Drawings

FIG. 1 shows the production of beta-alanine using FM 08.

FIG. 2 shows the production of 3-hydroxypropionic acid using FI 08.

FIG. 3 shows the production of beta-alanine by FA 11.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Wild-type P1 bacteriophage (Thomason LC, costatino N.2007.E. coli genome manipulation by. Current Protocols in Molecular Biology: 1.17.1-8) in the examples described below are publicly available from the institute of microbiology, a national academy of sciences, and the biomaterial is used only for repeating the experiments related to the present invention and is not used for other purposes.

In the following examples, E.coli BW25113(Datsenko KA, Wanner BL. one-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products. Proc. Natl. Acad. Sci. U.S.A.2000; 97 (12): 6640-6645.) is a non-pathogenic bacterium with clear genetic background, short generation time, easy culture and low cost of culture medium raw materials. Coli BW25113 is publicly available from the institute of microbiology, academy of sciences, and this biomaterial is only used for repeating the relevant experiments of the present invention, and is not used for other purposes.

Example 1 construction of recombinant E.coli engineering Strain FM07

This example prepared a basic strain FM07 that was used to prepare a strain that produced beta-alanine and 3-hydroxypropionic acid, as follows, using primers as shown in Table 1.

(1) Fatty acid degradation transcription factor fadR knockout.

The fadR gene of Escherichia coli BW25113 is knocked out from Escherichia coli BW25113 to obtain a mutant FM01 of Escherichia coli BW25113, and the specific steps are as follows:

(1-a) preparing a P1 bacteriophage containing an E.coli gene fragment having a fadR knock-out property.

The Escherichia coli gene fragment containing the fadR knockout character comes from an Escherichia coli strain JW1176, the strain is a W3110 series strain containing the fadR knockout character, JW1176 is a product of national institute of genetics (NIG, Japan) in which the gene fadR encoding a fatty acid degradation transcription factor is replaced with a kanamycin resistance gene (about 1300bp) having FRT sites at both ends to knock out the fadR gene (Baba T, Ara T, et al. construction of Escherichia coli K-12in-frame, single-gene knock out variants: the Keio collection. mol.Syst. biol. 2006; 2: 2006.0008.). The P1 phage was prepared as follows: the JW1176 strain is inoculated to the CaCl containing 5mmol/L after being cultured at 37 ℃ overnight₂And 0.1% glucose in LB medium, culturing at 37 deg.C for 1 hr, adding wild type P1 bacteriophage, and culturing for 1-3 hr. Adding a few drops of chloroform, culturing for a few minutes, centrifuging and taking the supernatant to obtain the phage P1vir fade R containing the escherichia coli gene fragment with the fade-out character.

(1-b) construction of E.coli strain FM01-Kan using P1 phage transduction:

escherichia coli BW25113 (recipient bacterium) cultured overnight was centrifuged at 10000g (1.5 mL) of the bacterial solution for 2 minutes, and then 0.75mL of a P1 salt solution (water as a solvent and 10mM CaCl as a solute) was added₂And 5mM MgSO₄) BW25113 bacterial cells were resuspended, 100. mu.L of phage P1vir fade R was mixed with 100. mu.L of BW25113 cell suspension, incubated at 37 ℃ for 30min, then 1mL of LB medium and 200. mu.L of 1mol/L sodium citrate were added, further culture was continued at 37 ℃ for 1h, the cells were collected by centrifugation, resuspended in 100. mu.L of LB medium, spread on a kanamycin-containing LB plate (kanamycin concentration was 50. mu.g/mL), cultured overnight at 37 ℃, colonies were selected, and PCR-amplified and identified with fade-IF/fade-IR primers (1700 bp band amplified was positive), and the selected positive colonies were designated as FM 01-Kan.

(1-c) Elimination of resistance:

the pCP20 plasmid (Clontech) was transformed into FM01-Kan by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plate containing ampicillin, clones were selected to obtain recombinant E.coli FM01-Kan/pCP20 containing the plasmid pCP 20. After culturing in LB medium containing ampicillin resistance at 30 ℃, spreading on an un-resistant LB plate and culturing overnight at 43 ℃, selecting clones, amplifying and identifying by using fadR-IF/fadR-IR primer PCR (amplified 400bp target band is positive), selecting positive clones and naming the positive clones as FM 01.

Among them, FM01 is a strain in which the fadR gene of the fatty acid-degrading transcription factor of Escherichia coli BW25113 was deleted. In Escherichia coli BW25113, the fadR gene encodes the protein shown in SEQ ID No.2, and the coding sequence of the fadR gene is shown in SEQ ID No. 1. The fadR-IF/fadR-IR amplified a fragment of about 400bp from the genomic DNA of FM01 and a fragment of about 1100bp from the genomic DNA of E.coli BW 25113. Wherein the fadR-IF primer binding site and the fadR-IR primer binding site are the upstream region and the downstream region of the fadR gene of Escherichia coli BW25113, respectively. Sequencing analysis results show that the genome of FM01 has no fadR gene, and FM01 is a mutant of Escherichia coli BW25113 obtained by knocking out the fadR gene of Escherichia coli BW 25113.

(2) Expression of the fadL gene is enhanced by promoter replacement.

Starting from recombinant strain FM01, replacing fadL gene promoter in the strain with Escherichia coli constitutive promoter P_CPA1Obtaining recombinant Escherichia coli FM02, which comprises the following steps:

(2-a) preparation of host bacteria containing pKD46 plasmid:

the FM01 strain obtained in the previous step was transformed with pKD46 plasmid (Clontech) by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plates containing ampicillin, clones were selected to obtain recombinant E.coli FM01/pKD46 containing plasmid pKD 46. After the induction of arabinose, the recombinant Escherichia coli FM01/pKD46 expresses 3 recombinant proteins of lambda phage, and the host bacteria have the capacity of homologous recombination. FM01/pKD46 competent cells were then prepared by 10% glycerol wash.

(2-b) preparation of plasmid for amplifying replacement promoter targeting gene fragment:

the nucleotide sequence of the CPA1-Lox66-Kan-Lox71 fragment is shown in SEQ ID No. 3. CPA1-Lox66-Kan-Lox71 contains: A. constitutive promoter P_CPA1The sequence, the nucleotide sequence of which is 1443-1622 of SEQ ID No.3, and the B-kanamycin resistance gene (LOXP-kan-LOXP) with a LOXP flank, the nucleotide sequence of which is 21-1433 of SEQ ID No. 3. CPA1-Lox66-Kan-Lox71 sequence was synthesized by whole gene (Nanjing Kinshiri Biotech Co., Ltd.)) And ligated to a pUC57 vector to obtain a recombinant vector pUC 57-9K.

(2-c) targeting fragment fadLup-kan-P_CPA1Preparation of fade down:

using pUC57-9K as a template, and adopting a primer fadL-PF/fadL-PR to amplify fadLup-kan-P_CPA1A fadLdown fragment, fadLup-kan-P_CPA1The sequence of the fadLdown fragment is SEQ ID No.4 in a sequence table, the fragment contains (a) fadLup of a promoter upstream homology arm of fadL gene, and the nucleotide sequence of the fragment is 1 st to 51 th sites of SEQ ID No. 4; (b) a kanamycin resistance gene (LOXP-kan-LOXP) with a LOXP flank, the nucleotide sequence of which is 52 to 1492 th of SEQ ID No. 4; (c) e.coli constitutive promoter P_CPA1The nucleotide sequence is position 1493-1670 of SEQ ID No. 4; (d) the nucleotide sequence of the fadL gene downstream homology arm fadLdown of the promoter is 1671-1722 th site of SEQ ID No. 4.

(2-d) homologous recombination:

mixing the fadLup-kan-P_CPA1Electro-transferring the-fadLdown fragment into the FM01/pKD46 competent cells prepared in (2-a), placing the cells on LB plate containing kanamycin (50. mu.g/ml) overnight at 37 ℃, selecting clones, performing PCR amplification identification by using fadL-PIF/fadL-PIR primers (the amplified band with about 2000bp is positive, the amplified band with about 400bp is negative), and selecting positive clones which are named as FM 02-kan. Wherein the primer binding positions are respectively the upstream and downstream regions of the fadL gene promoter of Escherichia coli BW 25113. Sequencing analysis showed that the genome of FM02-kan contained fadLup-kan-P from step (2-c)_CPA1-a fadLdown fragment.

(2-e) Elimination of resistance:

the pCP20 plasmid (Clontech) was transformed into FM02-Kan by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plate containing ampicillin, clones were selected to obtain recombinant E.coli FM02-Kan/pCP20 containing the plasmid pCP 20. Culturing in LB culture medium containing ampicillin resistance at 30 deg.C, spreading on nonresistant LB plate at 43 deg.C, culturing overnight, selecting clone, performing PCR amplification identification of fadL-PIF/fadL-PIR primer (amplified to obtain about 600bp target band as positive, amplified to obtain about 2000bp or 400bp target band as negative), selecting positive clone, and naming as FM 02.

Wherein FM02 is obtained by replacing the promoter of the fadL gene of FM01 with a constitutive promoter P_CPA1The strain of (1). In FM01, fadL gene encodes the protein shown in SEQ ID No.6, and fadL gene encoding sequence is shown in SEQ ID No. 5. Sequencing analysis results show that the fadL gene promoter on the genome of FM02 is replaced by a constitutive promoter P_CPA1Expression of the fadL Gene is represented by P_CPA1And (5) starting.

(3) Expression of the fadD gene is enhanced by promoter replacement.

Starting from recombinant strain FM02, the promoter of fatty acyl-CoA synthase fadD gene in the strain is replaced by Escherichia coli constitutive promoter P_CPA1Obtaining recombinant Escherichia coli FM03, which comprises the following steps:

(3-a) preparation of host bacteria containing pKD46 plasmid:

according to the method of step (2), pKD46 plasmid is transformed into the FM02 strain obtained in the last step, so as to obtain recombinant Escherichia coli FM02/pKD46 containing plasmid pKD46, and then FM02/pKD46 competent cells are prepared.

(3-b) targeting fragment fadDup-kan-P_CPA1Preparation of fadDdown:

using pUC57-9K in step (2) as a template, and amplifying fadDup-kan-P by using a primer fadD-PF/fadD-PR_CPA1fadDdown fragment, fadDup-kan-P_CPA1The sequence of the fadDdown fragment is SEQ ID No.7 in the sequence table, the fragment contains (a) fadDup of a promoter upstream homology arm of a fadD gene, and the nucleotide sequence of the fragment is 1 st to 51 th sites of the SEQ ID No. 7; (b) a kanamycin resistance gene (LOXP-kan-LOXP) with a LOXP flank, the nucleotide sequence of which is 52 to 1492 th of SEQ ID No. 7; (c) e.coli constitutive promoter P_CPA1The nucleotide sequence is position 1493-1670 of SEQ ID No. 7; (d) the nucleotide sequence of the homologous arm fadDdown of the fadD gene downstream of the promoter is 1671-1722 th site of SEQ ID No. 7.

(3-c) homologous recombination:

the fadDup-kan-P is added_CPA1Electro-transfer of the fadDdown fragment into the FM02/pKD46 competent cells prepared in (3-a) at 37 ℃ overnight on LB plates containing kanamycin (50. mu.g/ml)Selecting clones, carrying out PCR amplification identification by using fadD-PIF/fadD-PIR primers (the amplified 2000bp target band is positive, the amplified 400bp target band is negative), and selecting positive clones to be named as FM 03-kan. Wherein the primer binding positions are the upstream and downstream regions of the promoter of the fadD gene of Escherichia coli BW25113, respectively. Sequencing analysis showed that FM03-kan contains fadDup-kan-P of step (3-b) on the genome_CPA1-a fadDdown fragment.

(3-d) Elimination of resistance:

the kanamycin resistance of FM03-kan is eliminated by utilizing pCP20 plasmid according to the method of the step (2), the fadD-PIF/fadD-PIR primer is utilized for PCR amplification identification (the amplified band with about 600bp is positive, the amplified band with about 2000bp or 400bp is negative), and the selected positive clone is named as FM 03.

Wherein FM03 is obtained by replacing fadD gene promoter of FM02 with constitutive promoter P_CPA1The strain of (1). In FM02, the fadD gene encodes the protein shown in SEQ ID No.9, and the coding sequence of the fadD gene is shown in SEQ ID No. 8. Sequencing analysis results show that the fadD gene promoter on the genome of FM03 is replaced by a constitutive promoter P_CPA1Expression of the fadD Gene from P_CPA1And (5) starting.

(4) The expression of sthA gene was enhanced by promoter substitution.

Starting from recombinant strain FM03, the promoter of sthA gene of fatty acyl-CoA synthase in the strain is replaced by a constitutive promoter P of Escherichia coli_CPA1Obtaining recombinant Escherichia coli FM04, which comprises the following steps:

(4-a) preparation of host bacteria containing pKD46 plasmid:

according to the method of step (2), pKD46 plasmid is transformed into the FM03 strain obtained in the last step, so as to obtain recombinant Escherichia coli FM03/pKD46 containing plasmid pKD46, and then FM03/pKD46 competent cells are prepared.

(4-b) targeting fragment sthAUp-kan-P_CPA1Preparation of sthAdown:

using pUC57-9K of step (2) as a template, and amplifying sthAUp-kan-P by using primers sthA-PF/sthA-PR_CPA1The sthAldown fragment, sthApp-kan-P_CPA1Of the sthAdown fragmentThe sequence is SEQ ID No.10 in the sequence table, the fragment contains (a) a promoter upstream homology arm fadDup of sthA gene, and the nucleotide sequence is 1-51 of the SEQ ID No. 10; (b) a kanamycin resistance gene (LOXP-kan-LOXP) with a LOXP flank, the nucleotide sequence of which is 52 to 1492 th of SEQ ID No. 10; (c) e.coli constitutive promoter P_CPA1The nucleotide sequence is position 1493-1670 of SEQ ID No. 10; (d) the nucleotide sequence of the promoter downstream homology arm fadDdown of sthA gene is 1671-1722 th site of SEQ ID No. 10.

(4-c) homologous recombination:

mixing the above sthAUp-kan-P_CPA1The sthAdown fragment was electroporated into the FM03/pKD46 competent cells prepared in (4-a), and on LB plate containing kanamycin (50. mu.g/ml) overnight at 37 ℃, clones were selected, identified by PCR amplification using sthA-PIF/sthA-PIR primers (about 2000bp band amplified was positive, about 400bp band amplified was negative), and the selected positive clones were named as FM 04-kan. Wherein the primer binding positions are the upstream and downstream regions of the promoter of sthA gene of Escherichia coli BW25113, respectively. Sequencing analysis showed that the genome of FM04-kan contained sthAUp-kan-P of step (4-b)_CPA1-sthAdown fragment.

(4-d) Elimination of resistance:

the kanamycin resistance of FM04-kan was eliminated by the pCP20 plasmid according to the method of step (2), and the positive clone was selected and named as FM04 by PCR amplification and identification using sthA-PIF/sthA-PIR primers (the band of about 600bp amplified was positive, and the band of about 2000bp or 400bp amplified was negative).

Wherein FM04 is obtained by replacing sthA gene promoter of FM03 with constitutive promoter P_CPA1The strain of (1). In FM03, the sthA gene encodes the protein shown in SEQ ID No.12, and the coding sequence of sthA gene is shown in SEQ ID No. 11. Sequencing analysis results show that the promoter of sthA gene on the genome of FM04 is replaced by a constitutive promoter P_CPA1Expression of sthA Gene from P_CPA1And (5) starting.

(5) Knock-out of β -ketoacyl-ACP synthase II gene fabF.

Starting from the recombinant bacterium producing FM04, the fabF gene of FM04 is knocked out to obtain FM05, and the specific steps are as follows:

(5-a) preparing a P1 phage containing an E.coli gene fragment having a fabF knockout trait.

The Escherichia coli gene fragment containing the fabF knockout trait is derived from Escherichia coli strain JW1081, and JW1081 is a product of the Japanese national institute of genetics (NIG, Japan). Replacing the JW1176 strain with a strain JW1081 according to the P1 phage preparation method in the step (1) to obtain a phage P1vir fabF containing the escherichia coli gene fragment with the fabF knockout character.

(5-b) construction of E.coli strain FM05-Kan using P1 phage transduction:

replacing the Escherichia coli BW25113 with FM04 obtained in the step (4) according to the method of the step (1), carrying out PCR amplification identification by using fabF-IF/fabF-IR primers (the target band of about 1700bp is amplified to be positive), and selecting a positive clone to be named as FM 05-Kan.

(5-c) Elimination of resistance:

replacing FM01-Kan with FM05-Kan according to the method of the step (1), eliminating the kanamycin resistance of the strain, carrying out PCR amplification identification by using fabF-IF/fabF-IR primers (the amplified 400bp target band is positive), and selecting a positive clone to be named as FM 05.

Among them, FM05 is a strain in which the fabF gene of FM04 was deleted. In FM04, the fabF gene encodes the protein shown in SEQ ID No.14, and the coding sequence of the fabF gene is shown in SEQ ID No. 13. fabF-IF/fabF-IR amplified a fragment of about 400bp from genomic DNA of FM05 and a fragment of about 1600bp from genomic DNA of FM 04. Wherein the binding positions of the fabF-IF primer and the fabF-IR primer are the upstream region and the downstream region of the fabF gene of Escherichia coli BW25113, respectively. Sequencing analysis results show that the genome of FM05 has no fabF gene, and FM05 is a strain obtained by knocking out the fabF gene of FM 04.

(6) Knock-out of β -ketoacyl-ACP synthase III gene fabH.

Starting from the recombinant bacterium producing FM05, the fabH gene of FM05 is knocked out to obtain FM06, and the specific steps are as follows:

(6-a) preparing a P1 phage containing an E.coli gene fragment having a fabH knock-out property.

The E.coli gene fragment containing the fabH knockout trait is derived from an E.coli strain JW1077, and JW1077 is a product of the national institute of genetics (NIG, Japan). Replacing the JW1176 strain with a JW1077 strain according to the P1 phage preparation method in the step (1) to obtain a phage P1vir fabH containing the escherichia coli gene fragment with the fabH knockout character.

(6-b) construction of E.coli strain FM06-Kan using P1 phage transduction:

replacing the Escherichia coli BW25113 with the FM05 obtained in the step (4) according to the method in the step (1), carrying out PCR amplification identification by using fabH-IF/fabH-IR primers (the target band of 1700bp is amplified to be positive), and selecting a positive clone to be named as FM 06-Kan.

(6-c) Elimination of resistance:

replacing FM01-Kan with FM06-Kan according to the method of the step (1), eliminating the kanamycin resistance of the strain, carrying out PCR amplification identification by using fabH-IF/fabH-IR primers (the amplified 400bp target band is positive), and selecting a positive clone to be named as FM 06.

Among them, FM06 is a strain in which the fabH gene of FM05 was deleted. In FM05, the fabH gene encodes the protein shown in SEQ ID No.16, and the coding sequence of the fabH gene is shown in SEQ ID No. 15. fabH-IF/fabH-IR amplified a fragment of about 400bp from genomic DNA of FM06 and a fragment of about 1400bp from genomic DNA of FM 05. Wherein the binding positions of the fabH-IF primer and the fabH-IR primer are the upstream region and the downstream region of the fabH gene of Escherichia coli BW25113, respectively. Sequencing analysis results show that the genome of FM06 has no fabH gene, and FM06 is a strain obtained by knocking out the fabH gene of FM 05.

(7) Expression of the atoS and atoC genes is enhanced by promoter replacement.

Starting from recombinant strain FM06, the promoter of short-chain fatty acid degradation regulatory gene cluster atoSC (the gene cluster contains the atoS gene and the atoC gene) in the strain is replaced by an Escherichia coli constitutive promoter P_CPA1Obtaining recombinant Escherichia coli FM07, which comprises the following steps:

(7-a) preparation of host bacteria containing pKD46 plasmid:

according to the method of step (2), pKD46 plasmid is transformed into the FM06 strain obtained in the last step, so as to obtain recombinant Escherichia coli FM06/pKD46 containing plasmid pKD46, and then FM06/pKD46 competent cells are prepared.

(7-b) targeting fragment atoSCup-kan-P_CPA1-preparation of atoSCdown:

amplifying atoSCup-kan-P by using pUC57-9K in the step (2) as a template and adopting a primer atoSC-PF/atoSC-PR_CPA1-atoSCdown fragment, atoSCup-kan-P_CPA1-the sequence of the atoSCdown fragment is SEQ ID No.17 of the sequence listing, which fragment contains (a) the promoter upstream homology arm atoSCup of the atoSC gene cluster, the nucleotide sequence of which is position 1-51 of SEQ ID No. 17; (b) a kanamycin resistance gene (LOXP-kan-LOXP) with a LOXP flank, the nucleotide sequence of which is 52 to 1492 th of SEQ ID No. 17; (c) e.coli constitutive promoter P_CPA1The nucleotide sequence is position 1493-1670 of SEQ ID No. 17; (d) the nucleotide sequence of the homology arm atoSCdown at the downstream of the promoter of the atoSC gene cluster is 1671-1722 th site of SEQ ID No. 17.

(7-c) homologous recombination:

mixing the above atoSCup-kan-P_CPA1The atoSCdown fragment was electroporated (7-a) into the prepared FM06/pKD46 competent cells, and on LB plate containing kanamycin (50. mu.g/ml) overnight at 37 ℃, clones were selected, identified by PCR amplification using the atoSC-PIF/atoSC-PIR primers (2000 bp band amplified was positive and 400bp band amplified was negative), and the selected positive clones were named FM 07-kan. Wherein the primer binding positions are the upstream and downstream regions of the promoter of the atoSC gene cluster of Escherichia coli BW25113, respectively. Sequencing analysis showed that FM07-kan contained the atoSCup-kan-P of step (7-b) on its genome_CPA1-an atoSCdown fragment.

(7-d) Elimination of resistance:

the kanamycin resistance of FM07-kan was eliminated by the pCP20 plasmid according to the method of step (2), and the positive clone was selected and named as FM07 by PCR amplification identification using the primers atoSC-PIF/atoSC-PIR (the amplified band of about 600bp was positive and the amplified band of about 2000bp or 400bpbp was negative).

Wherein FM07 isReplacement of the promoter of the atoSC Gene Cluster of FM06 with the constitutive promoter P_CPA1The strain of (1). In FM06, the atoS gene in the atoSC gene cluster encodes the protein shown in SEQ ID No.19, the coding sequence of the atoS gene is shown in SEQ ID No.18, the atoC gene encodes the protein shown in SEQ ID No.21, and the coding sequence of the atoC gene is shown in SEQ ID No. 20. Sequencing analysis results showed that the promoter replacement of the atoSC gene cluster on the genome of FM07 is for constitutive promoter P_CPA1The expression of the atoS and atoC genes in the atoSC gene cluster is determined by P_CPA1And (5) starting.

TABLE 1 list of primer sequences used in example 1

Example 2 preparation of Strain FM08 for beta-alanine production and beta-alanine production

Preparation of strain FM08 for producing beta-alanine (beta-alanine)

FM08 was prepared as follows, using primers as shown in Table 2.

(1) Construction of a plasmid expressing the malonyl-CoA reductase truncation gene mcrC of phomophilus thermoaminovorus (Chloroflexus aurantiacaus).

(1-a) PCR amplification of the mcrC gene.

The nucleotide sequence of the modified malonyl-CoA reductase truncation gene mcrC of the pyrenophora thermophila (Chloroflexus aurantiacus) is shown as SEQ ID No.22, and the protein shown as SEQ ID No.23 in the coding sequence table. The mcrC gene shown in SEQ ID No.22 was synthesized as a whole gene, and then ligated to a pUC57 vector using the Gibson assembly method (Gibson DG, Young L, et al. enzymatic assembly of DNA molecules up to segmented cloned kits. Nat. methods. 2009; 6 (5): 343-. The mcrC-F and the mcrC-R are used as primers, a vector pUC57-mcrC is used as a template, and high fidelity TransStart FastPfu DNA polymerase (Beijing all-type gold biotechnology Co., Ltd., product catalog is AP221) is used for PCR amplification to obtain an mcrC gene segment with correct sequence.

(1-b) constructing a recombinant expression vector containing the mcrC gene.

The vector pLB1a (vector pLB1a has the nucleotide sequence shown in SEQ ID No.24) is digested with Ncol and Xhol, and the vector large fragment LB1a-NX is recovered. The correct mcrC gene fragment obtained in (1-a) above was ligated to LB1a-NX fragment by Gibson assembly. Escherichia coli DH 5. alpha. competent cells (CD 201, catalog of Kyoto Seikagaku Kogyo Co., Ltd.) were transformed by the CaCl2 method. Spread on LB plates containing ampicillin, and cultured overnight at 37 ℃. Selecting clones, identifying with primers F105-F/mcrC-R, selecting positive clones with correct target fragment sequences, extracting plasmids, and naming the obtained positive recombinant plasmids as pLB1 a-mcrC.

(2) Construction of a plasmid expressing a Corynebacterium glutamicum (Corynebacterium glutamicum _ Glutamicum) acetyl-CoA carboxylase acc gene cluster.

The Corynebacterium glutamicum acetyl coenzyme A carboxylase acc gene cluster is synthesized in a whole gene and is connected to a pUC57 vector by a Gibson assembly method to obtain a vector pUC 57-acc. The nucleotide sequence of the acc gene cluster is shown in SEQ ID No. 25. Wherein the RBS1 site is positioned in front of the accBC gene and has the sequence of 2-7 sites of SEQ ID No. 25; the nucleotide sequence of accBC is 15-1790 of SEQ ID No.25, the amino acid sequence is SEQ ID No.26, the nucleotide sequence of accDA is 1805-3259 of SEQ ID No.25, the amino acid sequence is SEQ ID No.27, RBS2 is contained between accBC and accDA, and the sequence is 1792-1797 of SEQ ID No. 25. The acc-F and acc-R are used as primers, a vector pUC57-acc is used as a template, and high fidelity TransStart Fastpfu DNA polymerase is used for PCR amplification to obtain an acc gene fragment with a correct sequence.

Digesting the plasmid pLB1a-mcrC of the step (1) by Xhol and EcoRI to obtain a large fragment LB1 a-mcrC-XE. The above-mentioned acc gene fragment was ligated with LB1a-mcrC-XE fragment by Gibson assembly. Coli DH 5. alpha. competent cells were transformed by the CaCl2 method. Spread on LB plates containing ampicillin, and cultured overnight at 37 ℃. Selecting clones, identifying by using a primer acc-F/T58-R, selecting positive clones with correct target fragment sequences to extract plasmids, and naming the obtained positive recombinant plasmids as pLB1 a-mcrC.

(3) Construction of a plasmid expressing an outer membrane protein gene alkL gene for uptake of foreign alkanes into Marinobacter hydrocarbonoclasus.

The genomic DNA of Hydrocarbon-removed Hypsizygus marmoreus was extracted using a bacterial genome extraction kit (Tiangen Biochemical technology Co., Ltd., product catalog DP 302). The alkL gene fragment was amplified by PCR using the extracted Haemophilus hydrocarbon-free genomic total DNA as a template and a primer alkL-F/alkL-R, while introducing the RBS sequence into the primer. The vector pLB1a-mcrC-acc obtained in step (2) was digested with EcoRI and Pstl to obtain large fragment LB1 a-mcrC-acc-EP. The above alkL gene fragment was ligated with LB1a-mcrC-acc-EP fragment by Gibson assembly. Transforming Escherichia coli DH5 alpha, identifying with primer alkL-F/T58-R, selecting positive clone extraction plasmid with correct target fragment sequence, and naming the obtained positive recombinant plasmid as pLB1 a-mcrC-acc-alkL.

pLB 1-1 a-mcrC-acc-alkL contains mcrC gene shown in SEQ ID No.22, acc gene cluster shown in SEQ ID No.25 and DNA fragment shown in SEQ ID No.28, wherein, the 2 nd to 7 th positions of SEQ ID No.28 are RBS sequence, and the 15 th to 686 th positions of SEQ ID No.28 are alkL nucleotide sequence. pLB1a-mcrC-acc-alkL can express mcrC protein shown in SEQ ID No.23, accBC protein shown in SEQ ID No.26, accDA protein shown in SEQ ID No.27 and alkL protein shown in SEQ ID No. 29.

(4) Construction of a plasmid expressing the baat gene (puuE gene) of the beta-alanine aminotransferase gene of Escherichia coli (Escherichia coli).

Genomic DNA was extracted from E.coli, and the puuE gene fragment was amplified using the primers puuE-F/puuE-R. The vector pSB1s (the nucleotide sequence of the vector pSB1s is shown in SEQ ID No.30) is digested with Ncol and Xhol, and the vector large fragment SB1s-NX is recovered. The pUE gene fragment was ligated to the SB1s-NX fragment using Gibson assembly. Transforming Escherichia coli DH5 alpha, identifying with primer F105-F/puuE-R, selecting positive clone with correct target fragment sequence, extracting plasmid, and naming the obtained positive recombinant plasmid as pSB1 s-puuE.

(5) Construction of a plasmid expressing the glutamic acid dehydrogenase gene gdh gene (rocG gene) of Bacillus subtilis.

Genomic DNA was extracted from E.coli, and the rocG gene fragment was amplified with the primers rocG-F/rocG-R while introducing the RBS sequence in the primers. The pSB1s-puuE of the vector step (4) was digested with Xhol and Pstl to obtain large fragment SB1 s-puuE-XP. And (3) carrying out a connection reaction on the rocG gene fragment and the SB1s-puuE-XP fragment. Transforming Escherichia coli DH5 alpha, identifying with primer rocG-F/T-58, selecting positive clone with correct target fragment sequence, extracting plasmid, and naming the obtained positive recombinant plasmid as pSB1 s-puuE-rocG.

pSB1s-puuE-rocG contains the puuE gene shown in SEQ ID No.31 and a DNA fragment shown in SEQ ID No.33, wherein the 2 nd to 7 th positions of SEQ ID No.33 are sequences of RBS, and the 15 th to 1289 th positions of SEQ ID No.33 are sequences of rocG gene. pSB1s-puuE-rocG can express puuE protein shown in SEQ ID No.32 and rocG protein shown in SEQ ID No. 34.

(6) Construction of recombinant E.coli FM 08.

Competent cells were prepared from the strain FM07 of example 1, and pLB1a-mcrC-acc-alkL and pSB1s-puuE-rocG prepared in the above procedure were introduced into FM 07. Spread on LB plates containing streptomycin and ampicillin, and cultured overnight at 37 ℃. A positive clone containing pLB1a-mcrC-acc-alkL and pSB1s-puuE-rocG was selected and named FM 08.

FM08 is a strain obtained by transforming Escherichia coli BW25113 into the following (a1) - (a 12):

(a1) knocking out a fadR gene of a fatty acid degradation transcription factor;

(a2) replacement of fadL Gene promoter with E.coli constitutive promoter P_CPA1；

(a3) Replacement of fadD Gene promoter with E.coli constitutive promoter P_CPA1；

(a4) Replacement of sthA Gene promoter with E.coli constitutive promoter P_CPA1；

(a5) Knocking out a fabF gene of a beta-ketoacyl-ACP synthase II gene;

(a6) knocking out a fabH gene of a beta-ketoacyl-ACP synthase III gene;

(a7) replacement of the atoSC Gene Cluster promoter with a constitutive promoter of E.coliSeed P_CPA1；

(a8) Introducing a malonyl coenzyme A reductase truncated gene mcrC gene;

(a9) introducing an acetyl coenzyme A carboxylase acc gene cluster;

(a10) introducing exogenous alkane to take in outer membrane protein gene alkL gene;

(a11) introducing a PUuE gene of a beta-alanine aminotransferase gene;

(a12) the glutamate dehydrogenase gene rocG was introduced.

Preparing strain FM07 into competent cells, and extracting plasmids pSB1s and pLB1a with CaCl₂FM07 was introduced. Spread on LB plates containing streptomycin and ampicillin, and cultured overnight at 37 ℃. Clones containing plasmids pSB1s and pLB1a were selected and designated FM00 as a control.

TABLE 2 list of primer sequences used in example 2

Preparation of di, beta-alanine

1. Preparation of the culture Medium

A culture medium: the culture medium A is a sterile culture medium consisting of a solute and a solvent, the solvent is water, and the solute and the concentration thereof are respectively as follows: 25mM NaHPO₄，25mM KH₂PO₄，50mM NH₄Cl，5mM Na₂SO₄，2mM MgSO₄0.5 percent of glycerin by volume percentage, 0.5 percent of yeast powder by mass percentage and 50 mu M FeCl₃，20μM CaCl₂，10μM MnCl₂，10μM ZnSO₄，2μM CoCl₂，2μM NiCl₂，2μM Na₂MO₄，2μM Na₂SeO₃And 2 μ M H₃BO₃。

B, culture medium: the B culture medium is a sterile culture medium obtained by adding palmitic acid, polyoxyethylene ether Brij58 emulsifier, Biotin and vitamin B6 into the A culture medium, wherein the mass percent concentration of the palmitic acid is 0.5%, the mass percent concentration of the polyoxyethylene ether Brij58 emulsifier is 0.2%, the mass percent concentration of the Biotin is 40mg/L, and the concentration of the vitamin B6 is 10 mg/L.

C, culture medium: the C culture medium is prepared by adding palmitic acid, polyoxyethylene ether Brij58 emulsifier, Biotin, NaHCO into the A culture medium₃Vitamin B6 and glutamic acid, wherein the mass percent concentration of palmitic acid is 1%, the mass percent concentration of polyoxyethylene ether Brij58 emulsifier is 0.2%, the concentration of Biotin is 40mg/L, and the concentration of NaHCO is 40mg/L₃The concentration of (A) was 20mM, the concentration of vitamin B6 was 10mg/L, and the concentration of glutamic acid was 2 mM.

2. Preparation of beta-alanine

The experiment is repeated three times, and the specific steps repeated in each experiment are as follows:

2.1 culturing of the cells.

The strain FM08 obtained in the first step of overnight culture was cultured as follows: inoculating the strain into 20ml of A culture medium containing streptomycin and kanamycin (both the concentrations of the streptomycin and the kanamycin are 50mg/L) according to the inoculation amount of 1%, culturing at 37 ℃ for 12h, and collecting thalli; the collected cells were transferred to 20ml of B medium containing streptomycin and kanamycin (both at 50mg/L) and cultured at 37 ℃ for 6 hours to obtain a culture solution, and OD of the culture solution was₆₀₀And 6, adding arabinose into the culture solution to induce the arabinose to be 0.2 percent of the mass percentage concentration of the arabinose in the culture solution, culturing for 12 hours at 37 ℃, and collecting thalli to obtain the FM08 thalli.

FM00 was cultured in the above-described manner using medium A and medium B which did not contain streptomycin and kanamycin, to obtain FM00 cells.

2.2 Whole-cell catalytic production of beta-alanine.

30mg (i.e. 1X 10) collected in step 2.1 above¹¹cfu) dry weight of FM08 thallus is resuspended in a shake flask containing 20ml of C culture medium, after culturing for 24h at 37 ℃, the supernatant fluid is obtained by centrifugation and filtered by a 0.22 mu m filter, and the filtrate is the FM08 sample to be detected.

According to the method, FM08 is replaced by FM00 thallus, and other steps are not changed, so that the FM00 sample to be tested is obtained.

And (3) quantitatively analyzing the content of the beta-alanine in each sample to be detected by using the beta-alanine (Sigma, 05159-100G) as a standard sample and using an HPLC (high performance liquid chromatography) standard curve method (an external standard method).

The quantitative determination result is shown in FIG. 1, and the average content of beta-alanine in FM08 sample is 0.36g/L (i.e. 0.36 g/5X 10)¹²cfu), wherein the mass percentage concentration of the palmitic acid is 0.78%; the average content of beta-alanine in the FM00 sample to be detected is 0mg/L, and the mass percentage concentration of palmitic acid is 0.89%. The conversion rate of beta-alanine produced using FM08 with palmitic acid as a substrate was 16.36%, and beta-alanine could not be obtained using FM 00. It was shown that FM08 can be used to produce beta-alanine.

Example 3 preparation of Strain FI08 for production of 3-hydroxypropionic acid and production of 3-hydroxypropionic acid

Preparation of strain FI08 for producing 3-hydroxypropionic acid

FI08 was prepared as follows, and the primers used are shown in Table 3.

(1) Construction of a plasmid expressing a Corynebacterium glutamicum (Corynebacterium glutamicum _ Glutamicum) acetyl-CoA carboxylase acc gene cluster.

(1-a) extraction of Corynebacterium glutamicum genomic DNA and PCR amplification of the acc gene cluster.

Corynebacterium glutamicum genomic DNA was extracted using a bacterial genome extraction kit (Tiangen Biochemical technology Co., Ltd., catalog DP 302). Extracting corynebacterium glutamicum genome total DNA as a template, taking accBC-F and accL-R as primers, amplifying a gene segment accBC by using high-fidelity TransStart Fastpfu DNA polymerase PCR, and recovering a target segment by agarose gel electrophoresis. The gene fragment accDA is amplified by using TransStart Fastpfu DNA polymerase PCR (polymerase chain reaction) by using Corynebacterium glutamicum genome total DNA as a template and accL-F and accDA-R as primers, and the target fragment is recovered by agarose gel electrophoresis. Wherein the accDA-R primer is introduced with a Nhel site to facilitate the subsequent gene fragment insertion; the 3 'end of the accBC fragment and the 5' end of the accDA fragment were primed with the complementary sequence containing the RBS for the next round of splicing. And (3) further carrying out PCR amplification on an acc fragment with a full-length gene sequence by using a mixture of the accBC and the accDA as a template and the accBC-F and the accDA-R as primers, and recovering the target fragment by agarose gel electrophoresis.

(1-b) constructing a recombinant expression vector containing the acc gene.

The vector pSB1s (the nucleotide sequence of the vector pSB1s is shown in SEQ ID No.30) is digested with Ncol and Xhol, and the vector large fragment SB1s-NX is recovered. The acc fragment was ligated with SB1s-NX fragment by Gibson assembly. Coli DH 5. alpha. competent cells were transformed by the CaCl2 method. The suspension was spread evenly on LB plates containing streptomycin and cultured overnight at 37 ℃. Selecting clones, identifying clones capable of amplifying target fragments by using the primers F-105/accL-R, sequencing, selecting positive clones, extracting plasmids, and obtaining positive plasmids which are named as pSB1 s-acc. pSB1s-acc contains the DNA fragment shown in positions 15-3259 of SEQ ID No. 25.

(2) Construction of a plasmid expressing an outer membrane protein gene alkL gene for uptake of foreign alkanes into Marinobacter hydrocarbonoclasus.

Genomic DNA was extracted from marinobacter hydrocarbonoclavus, and the alkL gene fragment was amplified with primers alkL-F/alkL-R' while introducing the RBS sequence in the primers. The vector pSB1s-acc was digested with Nhel and Spel to obtain the large fragment SB1 s-acc-HS. The ligation reaction of the alkL fragment with the SB1s-acc-HS fragment was performed by Gibson assembly. Transforming Escherichia coli DH5 alpha, identifying with primer alkL-F/T-58, selecting positive clone with correct target fragment sequence, extracting plasmid, and naming the obtained positive recombinant plasmid as pSB1 s-acc-alkL.

pSB1s-acc-alkL contains a DNA fragment shown in SEQ ID No.25 from position 15 to 3259 and a DNA fragment shown in SEQ ID No.28, wherein position 2 to 7 of SEQ ID No.28 is the sequence of RBS and position 15 to 686 of SEQ ID No.28 is the nucleotide sequence of alkL. pSB1s-acc-alkL can represent the accBC protein shown in SEQ ID No.26, the accDA protein shown in SEQ ID No.27 and the alkL protein shown in SEQ ID No. 29.

(3) Construction of a plasmid expressing the malonyl-CoA reductase gene mcr of Thermophilus aurescens (Chloroflexus aurantiacus).

(3-a) PCR amplification of the mcr gene.

The nucleotide sequence of the engineered Thermopsis rhodochrous (CMoroflex aurantiacus) malonyl coenzyme A reductase gene mcr gene is shown as SEQ ID No.36, wherein the nucleotide sequence of the N-terminal domain of mcr is 1-1689 th site of SEQ ID No.36, the nucleotide sequence of the C-terminal domain of mcr is 1704 th-3749 th site of SEQ ID No.36, an RBS site is arranged between the N-terminal domain and the C-terminal domain, and the sequence is 1691 1696 th site of SEQ ID No. 36. The mcr gene sequence was synthesized by whole gene synthesis and ligated to pUC57 vector by Gibson assembly to obtain vector pUC 57-mcr. And using pUC57-mcr as a template and using a primer mcr-F/mcr-R for amplification to obtain an mcr gene fragment with a correct sequence.

(3-b) constructing a recombinant expression vector containing the mcr gene.

Carrying out agarose gel electrophoresis on the mcr gene fragment with the correct sequence obtained in the step (3-a), and recovering a target fragment; the vector pXB1k (the nucleotide sequence of the vector pXB1k is shown in SEQ ID No.35) is cut by Ncol and Xhol, and the vector large fragment XB1k-NX is recovered. The correctly sequenced mcr gene fragment obtained in (3-a) above was ligated to the XB1k-NX fragment by Gibson's assembly method. Coli DH 5. alpha. competent cells were transformed by the CaCl2 method. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. Selecting clones, identifying clones capable of amplifying target fragments by using a primer F-105/mcr-R, sequencing, selecting positive clones, extracting plasmids, and obtaining the positive plasmids which are named as pXB1 k-mcr.

pXB1k-mcr contains a DNA fragment shown in SEQ ID No.36 and can express mcr protein shown in SEQ ID No. 37.

(4) Construction of recombinant E.coli FM 08.

Competent cells were prepared from strain FM07 of example 1, plasmids pSB1s-acc-alkL and pXB1k-mcr were added with CaCl₂FM 07. Spread on LB plates containing streptomycin and kanamycin, and cultured overnight at 37 ℃. A positive clone containing pSB1s-acc-alkL and pXB1k-mcr was selected and named FI 08.

FI08 is a strain obtained by transforming Escherichia coli BW25113 into the following strains (b1) - (b 10):

(b1) knocking out a fadR gene of a fatty acid degradation transcription factor;

(b2) the fadL geneReplacement of the promoter with the constitutive promoter P of E.coli_CPA1；

(b3) Replacement of fadD Gene promoter with E.coli constitutive promoter P_CPA1；

(b4) Replacement of sthA Gene promoter with E.coli constitutive promoter P_CPA1；

(b5) Knocking out a fabF gene of a beta-ketoacyl-ACP synthase II gene;

(b6) knocking out a fabH gene of a beta-ketoacyl-ACP synthase III gene;

(b7) replacement of the atoSC Gene Cluster promoter with the E.coli constitutive promoter P_CPA1；

(b8) Introducing an acetyl coenzyme A carboxylase acc gene cluster;

(b9) introducing exogenous alkane to take in outer membrane protein gene alkL gene;

(b10) malonyl-coa reductase gene mcr gene.

Competent cells were prepared from strain FM07 of example 1, and plasmids pSB1s and pXB1k were introduced into FM07 by the CaCl2 method. Spread on LB plates containing streptomycin and ampicillin, and cultured overnight at 37 ℃. A clone containing plasmids pSB1s and pXB1k was selected and designated FC00 as a control.

TABLE 3 primer sequences List used in example 3

Preparation of di, 3-hydroxypropionic acid (3-HP)

1. Preparation of the culture Medium

D, culture medium: the medium D was a sterile medium obtained by adding palmitic acid and a polyoxyethylene ether Brij58 emulsifier to the medium A of example 2, wherein the mass percentage concentration of palmitic acid was 0.5%, and the mass percentage concentration of the polyoxyethylene ether Brij58 emulsifier was 0.2%.

E, culture medium: the E medium is the medium A of example 2 added with palmitic acid, polyoxyethylene ether Brij58 emulsifier, Biotin and NaHCO₃The obtained sterile culture medium is used for culturing a culture medium,wherein, the mass percent concentration of the palmitic acid is 1 percent, the mass percent concentration of the polyoxyethylene ether Brij58 emulsifier is 0.2 percent, the concentration of the Biotin is 40mg/L, and the concentration of NaHCO is 40mg/L₃Is 20 mM.

2. Preparation of 3-hydroxypropionic acid

2.1 culturing of the cells.

The strain FI08 obtained in the first step of overnight culture was cultured as follows: the strain was inoculated at 1% inoculum size into 20ml of the streptomycin-and-kanamycin-containing A medium of example 2 (both streptomycin and kanamycin are 50mg/L), cultured at 37 ℃ for 12 hours, and the cells were collected; the collected cells were transferred to 20ml of a D medium containing streptomycin and kanamycin (both at 50mg/L) and cultured at 37 ℃ for 6 hours to obtain a culture solution, and OD of the culture solution was₆₀₀6, arabinose was added to the culture solution to induce arabinose to a concentration of 0.2% by mass in the culture solution, and the mixture was cultured at 37 ℃ for 12 hours to collect cells, that is, FI08 cells.

According to the above method, FC00 was cultured in medium A and medium D which did not contain streptomycin or kanamycin, to obtain FC00 cells.

2.2 Whole-cell catalytic production of 3-hydroxypropionic acid.

30mg (i.e. 1X 10) collected in step 2.1 above¹¹cfu) dry weight FI08 thallus is resuspended in a shake flask containing 20ml E culture medium, after culturing for 24h at 37 ℃, the supernatant fluid is obtained by centrifugation and filtered by a 0.22 μm filter, and the filtrate is FI08 sample to be detected.

According to the method, FI08 is replaced by FC00 thallus, and other steps are not changed, so that the FC00 sample to be tested is obtained.

And (3) quantitatively analyzing the content of the 3-hydroxypropionic acid in each sample to be detected by using the 3-hydroxypropionic acid (TCI, H0297-10G) as a standard sample and using an HPLC (high performance liquid chromatography) method by using a standard curve method (an external standard method).

The quantitative determination result is shown in FIG. 2, the average content of 3-hydroxypropionic acid in the FI08 sample to be determined is 0.539g/L (i.e. 0.539 g/5X 10¹²cfu), palmitic acidThe mass percentage concentration is 0.81 percent; the average content of the 3-hydroxypropionic acid in the FC00 sample to be tested is 0g/L, and the mass percentage concentration of the palmitic acid is 0.91%. The conversion of 3-hydroxypropionic acid using FI08 and palmitic acid as a substrate was 28.37%, and 3-hydroxypropionic acid could not be obtained using FC 00. It is shown that 3-hydroxypropionic acid can be prepared using FI 08.

Example 4 preparation of Strain FA11 for production of beta-alanine and production of beta-alanine

Preparation of a Strain FA11 for producing beta-alanine

FA11 was prepared as follows, using primers as shown in Table 4.

(1) Knock-out of the glyoxylate pathway transcription repressor gene iclR.

An iclR gene of FM07 is knocked out from the recombinant strain FM07 in example 1 to obtain FA08, and the specific steps are as follows:

(1-a) preparing P1 phage containing E.coli gene fragment having iclR knockout trait.

The E.coli gene fragment containing the iclR knockout trait is derived from the E.coli strain JW3978, and JW3978 is a product of the Japanese national institute of genetics (NIG, Japan). The phage P1vir iclR containing the E.coli gene fragment with the iclR knock-out property was obtained by replacing the strain JW1176 with the strain JW3978 according to the P1 phage preparation method of step (1) in example 1.

(1-b) construction of E.coli strain FA08-Kan using P1 phage transduction:

escherichia coli BW25113 was replaced with the recombinant strain FM07 of example 1 by the method of step (1) of example 1, and identified by PCR amplification using iclR-IF/iclR-IR primers (1700 bp of band was amplified as positive), and the positive clone selected was named FA 08-Kan.

(1-c) Elimination of resistance:

the strain was verified for kanamycin resistance by replacing FM01-Kan with FA08-Kan according to the method of step (1) in example 1, identified by PCR amplification using iclR-IF/iclR-IR primers (400 bp bands were amplified as positive), and the positive clone was selected and named FA 08.

Wherein FA08 is the iclR gene knockout strain of FM07 in example 1. In FM07, the iclR gene encodes the protein shown in SEQ ID No.39, and the coding sequence of the iclR gene is shown in SEQ ID No. 38. iclR-IF/iclR-IR amplified an about 400bp fragment from genomic DNA of FA08 and an about 1200bp fragment from genomic DNA of FM 07. Wherein the iclR-IF and iclR-IR primer binding sites are the upstream region and the downstream region of the iclR gene of E.coli BW25113, respectively. Sequencing analysis results show that the genome of FA08 has no iclR gene, and FA08 is a strain obtained by knocking out the iclR gene of FM07 in example 1.

(2) Knock-out of alpha-ketoglutarate decarboxylase gene sucA.

Starting from FA08, the sucA gene of FA08 is knocked out to obtain FA09, and the specific steps are as follows:

(2-a) preparation of P1 phage containing E.coli gene fragment having sucA knockout trait.

The escherichia coli gene fragment containing the sucA knockout character is derived from an escherichia coli strain JW0715, and the JW0715 is a product of the national institute of genetics (NIG, Japan). The P1 phage preparation method in step (1) of example 1 was followed to replace JW1176 strain with JW0715 strain to obtain phage P1vir sucA containing the sucA knockout E.coli gene fragment.

(2-b) construction of E.coli strain FA09-Kan using P1 phage transduction:

coli BW25113 was replaced with FA08 by the method of step (1) in example 1, and identified by PCR amplification using sucA-IF/sucA-IR primers (1700 bp of the target band was amplified as positive), and the positive clone was selected and named FA 00-Kan.

(2-c) Elimination of resistance:

the strain was verified to be kana resistant by replacing FM01-Kan with FA09-Kan according to the method of step (1) in example 1, and then identified by PCR amplification using sucA-IF/sucA-IR primers (400 bp bands were amplified as positive), and the positive clone was selected and named FA 09.

Of these, FA09 was a knockout strain of the sucA gene of FA 08. In FA08, the sucA gene encodes a protein shown by SEQ ID No.41, and the coding sequence of the sucA gene is shown by SEQ ID No. 40. The sucA-IF/sucA-IR amplified from the genomic DNA of FA09 gave a fragment of about 400bp, and from the genomic DNA of FM08 gave a fragment of about 3200 bp. Wherein the sucA-IF primer binding site and the sucA-IR primer binding site are the upstream region and the downstream region of the sucA gene of Escherichia coli BW25113, respectively. Sequencing analysis results show that the genome of FA00 has no sucA gene, and FA09 is a strain obtained by knocking out the sucA gene of FA 08.

(3) The expression of the aceB gene and the aceA gene is enhanced by promoter replacement.

Starting from recombinant bacteria FA09, replacing the promoter of the glyoxylate pathway aceBA gene cluster (the gene cluster contains aceB gene and aceA gene) in the strain with escherichia coli constitutive promoter P_CPA1Obtaining recombinant Escherichia coli FA10, which comprises the following steps:

(3-a) preparation of host bacteria containing pKD46 plasmid:

according to the method of the step (2) of the example 1, the pKD46 plasmid is transformed into the FA09 strain obtained in the last step, so as to obtain the recombinant Escherichia coli FA09/pKD46 containing the plasmid pKD46, and then the FA09/pKD46 competent cells are prepared.

(3-b) targeting fragment aceBAup-kan-P_CPA1-preparation of aceBAdown:

aceBAup-kan-P was amplified using pUC57-9K of step (2) of example 1 as a template and aceBA-PF/aceBA-PR as a primer_CPA1-aceBAdown fragment, aceBAup-kan-P_CPA1The sequence of aceBAdown fragment is SEQ ID No.42 in the sequence table, the fragment contains (a) aceBAup of the upstream homologous arm of the promoter of aceBA gene cluster, and the nucleotide sequence is 1-51 th of SEQ ID No. 42; (b) a kanamycin resistance gene (LOXP-kan-LOXP) with a LOXP flank, the nucleotide sequence of which is 52 to 1492 th of SEQ ID No. 42; (c) e.coli constitutive promoter P_CPA1The nucleotide sequence is position 1493-1670 of SEQ ID No. 42; (d) the nucleotide sequence of the aceBAdown homologous arm of the promoter downstream of the aceBA gene cluster is 1671-1722 th site of SEQ ID No. 42.

(3-c) homologous recombination:

mixing the aceBAup-kan-P_CPA1Electrotransfer of aceBAdown fragment (3-a) to FA09/pKD prepared46 competent cells were cultured overnight at 37 ℃ on LB plates containing 50. mu.g/ml kanamycin to select clones, and the clones were identified by PCR amplification using aceBA-PIF/aceBA-PIR primers (amplified band of about 2000bp was positive and amplified band of about 400bp was negative), and the selected positive clones were named FA 10-kan. Wherein the primer binding positions are respectively the upstream and downstream regions of the promoter of the aceBA gene cluster of Escherichia coli BW 25113. Sequencing analysis showed that FA10-kan contains aceBAup-kan-P of step (3-b) on its genome_CPA1-aceBAdown fragment.

(3-d) Elimination of resistance:

the kanamycin resistance of FA10-kan was eliminated by the pCP20 plasmid according to the method of step (2) of example 1, and the positive clone was selected and named FA10 by PCR amplification identification using aceBA-PIF/aceBA-PIR primers (positive for about 600bp band amplified, negative for about 2000 or 400bp band amplified).

Wherein FA10 is the constitutive promoter P substituted by the promoter of the aceBA gene cluster of FA09_CPA1The strain of (1). In FA09, the aceB gene in the aceBA gene cluster encodes the protein shown in SEQ ID No.44, the coding sequence of the aceB gene is shown in SEQ ID No.43, the aceA gene encodes the protein shown in SEQ ID No.46, and the coding sequence of the aceA gene is shown in SEQ ID No. 45. The sequencing analysis result shows that the promoter of the aceBA gene cluster on the genome of FA10 is replaced by a constitutive promoter P_CPA1The expression of aceB gene and aceA gene in aceBA gene cluster is controlled by P_CPA1And (5) starting.

(4) Construction of a plasmid expressing the E.coli (Escherichia coli) aspartate aminotransferase gene aspC.

(4-a) extraction of E.coli genomic DNA and PCR amplification of the aspC gene.

Coli genomic DNA was extracted using a bacterial genome extraction kit (Tiangen Biochemical technology Ltd., product catalog DP 302). The extracted total DNA of the Escherichia coli genome is used as a template, aspC-F and aspC-R are used as primers, and a high fidelity TransStart FastPfu DNA polymerase (Beijing Quanzijin Biotechnology Co., Ltd., product catalog: AP221) is used for PCR amplification to obtain a gene fragment aspC with a correct sequence.

(4-b) construction of a recombinant expression vector containing the aspC gene.

The vector pLB1a (vector pLB1a has the nucleotide sequence shown in SEQ ID No.24) is digested with Ncol and Xhol, and the vector large fragment LB1a-NX is recovered. The gene fragment aspC with the correct sequence obtained in the above step was ligated to the LB1a-NX fragment by the Gibson assembly method. Coli DH 5. alpha. competent cells were transformed by the CaCl2 method. The suspension was spread on an LB plate containing ampicillin uniformly and cultured overnight at 37 ℃. Selecting clone, using primer F105-F/aspC-R to identify, selecting positive clone with correct target fragment sequence to extract plasmid, and naming the obtained positive recombinant plasmid as pLB1 a-aspC.

(5) Construction of a plasmid expressing the glutamate dehydrogenase gene gdhA of Escherichia coli (Escherichia coli).

Genomic DNA was extracted from E.coli, and the gdhA gene fragment was amplified using the primers gdhA-F/gdhA-R, while the RBS sequence was introduced into the primers. The vector pLB1a-aspC was digested with Xhol and Spel to obtain large fragment LB1 a-aspC-XP. The gdhA gene fragment and the LB1a-aspC-XP fragment were ligated by the Gibson assembly method. Transforming Escherichia coli DH5 alpha, identifying with primer gdhA-F/T58-R, selecting positive clone with correct target fragment sequence to extract plasmid, and naming the obtained positive recombinant plasmid as pLB1 a-aspC-gdhA.

(6) Construction of a plasmid expressing an outer membrane protein gene alkL gene for uptake of foreign alkanes into Marinobacter hydrocarbonoclasus.

Genomic DNA was extracted from marinobacter hydrocarbonoclavus, and the alkL gene fragment was amplified with the primer alkL-F '/alkL-R' while the RBS sequence was introduced into the primer. The vector pLB1a-aspC-gdhA was digested with Spel and EcoRI to obtain the large fragment LB1 a-aspC-gdhA-PE. The alkL gene fragment and LB1a-aspC-gdhA-PE fragment were ligated by Gibson assembly. Transforming Escherichia coli DH5 alpha, identifying with primer alkL-F/T58-R, selecting positive clone with correct target fragment sequence to extract plasmid, and naming the obtained positive recombinant plasmid as pLB1 a-aspC-gdhA-alkL.

pLB1a-aspC-gdhA-alkL contains aspC gene shown in SEQ ID No.47, gdhA gene shown in SEQ ID No.49 and DNA fragment (containing alkL gene) shown in SEQ ID No. 28. Wherein, the 2 nd to 7 th positions of SEQ ID No.49 are sequences of RBS, and the 15 th to 1358 th positions of SEQ ID No.49 are sequences of gdhA genes. pLB1a-aspC-gdhA-alkL can express aspC protein shown in SEQ ID No.48, gdhA protein shown in SEQ ID No.50 and alkL protein shown in SEQ ID No. 29.

(7) Construction of a plasmid expressing the panD gene of the L-aspartate-alpha-decarboxylase of Tribolium castaneum.

The L-aspartic acid-alpha-decarboxylase gene panD of the total gene synthesis Tribolium castaneum is connected to a pUC57 vector to obtain a vector pUC 57-panD. The nucleotide sequence of the panD gene is shown in SEQ ID No. 51. Using panD-F and panD-R as primers and pUC57-panD plasmid as template, panD gene fragment was amplified by high fidelity TransStart Fastpfu DNA polymerase PCR. The vector pXB1k (the nucleotide sequence of the vector pXB1k is shown in SEQ ID No.35) is cut by Ncol and Xhol, and the vector large fragment XB1k-NX is recovered. The panD gene fragment was ligated to the XB1k-NX fragment using the Gibson assembly method. Coli DH 5. alpha. was transformed, plated on LB plates containing kanamycin and cultured overnight at 37 ℃ to select clones. The primers F105-F/panD-R are used for identification, positive clones with correct target fragment sequences are selected, plasmids are extracted, and the obtained positive recombinant plasmids are named pXB1 k-panD. pXB1k-panD contains the panD gene shown by SEQ ID No.51 and can express the panD protein shown by SEQ ID No. 52.

(8) Construction of recombinant E.coli FA 11.

Competent cells were prepared from the strain FA10 obtained in step (3), and the plasmids pLB1a-aspC-gdhA-alkL and pXB1k-panD were transformed with FA10 by the CaCl2 method. Spread on LB plates containing ampicillin and kanamycin, and incubated overnight at 37 ℃. A positive clone containing pLB1a-aspC-gdhA-alkL and pXB1k-panD was selected and named FA 11.

FA11 is a strain obtained by transforming E.coli BW25113 as follows (c1) - (c 14):

(c1) knocking out a fadR gene of a fatty acid degradation transcription factor;

(c2) replacement of fadL Gene promoter with E.coli constitutive promoter P_CPA1；

(c3) Replacement of fadD Gene promoter with E.coli constitutive promoter P_CPA1；

(c4) Replacement of sthA Gene promoter with E.coli constitutive promoter P_CPA1；

(c5) Knocking out a fabF gene of a beta-ketoacyl-ACP synthase II gene;

(c6) knocking out a fabH gene of a beta-ketoacyl-ACP synthase III gene;

(c7) replacement of the atoSC Gene Cluster promoter with the E.coli constitutive promoter P_CPA1；

(c8) Knocking out a glyoxylate pathway transcription inhibitor gene iclR gene;

(c9) knocking out an alpha-ketoglutarate decarboxylase gene sucA gene;

(c10) replacement of aceBA gene cluster promoter with E.coli constitutive promoter P_CPA1；

(c11) Introducing an aspartate aminotransferase gene aspC gene;

(c12) introducing a glutamate dehydrogenase gene gdhA gene;

(c13) introducing exogenous alkane to take in outer membrane protein gene alkL gene;

(c14) the panD gene was introduced as an L-aspartate-. alpha. -decarboxylase gene.

Preparing competent cells from strain FA10, and extracting plasmids pLB1a and pXB1k with CaCl₂FA10 was transformed. Spread on LB plates containing ampicillin and kanamycin, and incubated overnight at 37 ℃. A positive clone containing pLB1a and pXB1k was selected and named FA 00.

TABLE 4 list of primer sequences used in example 4

Preparation of di, beta-alanine

1. Preparation of the culture Medium

F, culture medium: the F medium was a sterile medium obtained by adding palmitic acid, polyoxyethylene ether Brij58 emulsifier and vitamin B6 to the A medium of example 2, wherein the mass percentage concentration of palmitic acid was 0.5%, the mass percentage concentration of polyoxyethylene ether Brjj58 emulsifier was 0.2%, and the concentration of vitamin B6 was 10 mg/L.

G medium: the G medium was a sterile medium obtained by adding palmitic acid, polyoxyethylene ether Brij58 emulsifier, vitamin B6 and glutamic acid to the A medium of example 2, wherein the concentration of palmitic acid was 1% by mass, the concentration of polyoxyethylene ether Brij58 emulsifier was 0.2% by mass, the concentration of vitamin B6 was 10mg/L, and the concentration of glutamic acid was 2 mM.

2. Preparation of beta-alanine

2.1 culturing of the cells.

The strain FA11 obtained in the first step of overnight culture was cultured as follows: the strain was inoculated at 1% inoculum size into 20ml of A medium containing streptomycin and kanamycin (both at 50mg/L) of example 2, cultured at 37 ℃ for 12 hours, and the cells were collected; the collected cells were transferred to 20ml of F medium containing streptomycin and kanamycin (both at 50mg/L) and cultured at 37 ℃ for 6 hours to obtain a culture solution, the OD of which was₆₀₀And 6, adding arabinose into the culture solution to induce the arabinose to be 0.2 percent of the mass percent concentration in the culture solution, culturing for 12 hours at 37 ℃, and collecting thalli to obtain the FA11 thalli.

FA00 cells were obtained by culturing FA00 in the presence of A medium and F medium, which do not contain streptomycin and kanamycin, as described above.

2.2 Whole-cell catalytic production of beta-alanine.

30mg (i.e. 1X 10) collected in step 2.1 above¹¹cfu) dry weight of FA11 thallus is resuspended in a shake flask containing 20ml of G culture medium, cultured at 37 ℃ for 24h, centrifuged, taken supernatant and filtered by a 0.22 mu m filter to obtain filtrate, namely the FA11 sample to be detected.

According to the method, FA11 is replaced by FA00 thallus, and other steps are not changed, so that the FA00 sample to be detected is obtained.

The quantitative determination result is shown in FIG. 3, and the average content of beta-alanine in FA11 sample is 4.2g/L (i.e. 4.2g/5 × 10)¹²cfu), wherein the mass percent concentration of the palmitic acid is 0.31%; the average content of beta-alanine in the FA00 sample to be detected is 0g/L, and the mass percentage concentration of palmitic acid is 0.90%. The conversion rate of producing beta-alanine by using FA11 and palmitic acid as a substrate was 60.87%, and beta-alanine could not be obtained by using FA 00. It was shown that FA11 can be used to produce beta-alanine.

Claims

1. The construction method of the recombinant bacterium comprises the steps of carrying out the total transformation of A1-A8 and B1-B6 on a recipient bacterium to obtain the recombinant bacterium;

a1, knocking out fatty acid degradation transcription factor fadR gene of the recipient bacterium, or inhibiting the expression of the fadR gene or inhibiting the activity of protein encoded by the fadR gene;

a4, knocking out the iclR gene of the recipient bacterium or inhibiting the expression of the iclR gene or inhibiting the activity of the protein encoded by the iclR gene;

the gene in the short-chain fatty acid degradation pathway is B5a or B5B:

B5B, short chain fatty acid degradation gene cluster atoDAEB gene cluster;

b6, increasing the content of protein coded by aceBA gene cluster in glyoxylate pathway in the recipient bacterium or enhancing the activity of protein coded by aceBA gene cluster in glyoxylate pathway;

2. The method of claim 1, wherein: the recipient bacterium is escherichia coli;

and/or the alkL gene is derived from Haemophilus hydrocarbonoclosticus (Marinobacter hydrocarbonoclosticus) or/and Pseudomonas putida (Pseudomonas putida);

the gene in the L-aspartic acid synthesis pathway is a gene aspC gene which is derived from Escherichia coli (Escherichia coli) or/and Alcaligenes eutrophus (Ralstonia eutropha) or/and Pseudomonas aeruginosa (Pseudomonas aeruginosa) and codes aspartate aminotransferase, a gene aspDH gene which codes L-aspartate dehydrogenase or/and a gene aspA gene which codes aspartate lyase;

the panD gene is derived from Tribolium castaneum or/and Escherichia coli or/and Bacillus subtilis.

3. The method of claim 2, wherein: the fadR gene codes the protein shown by SEQ ID No. 2in the sequence table;

the fabF gene codes a protein shown by SEQ ID No.14 in a sequence table;

the fabH gene codes a protein shown by SEQ ID No.16 in a sequence table;

the iclR gene codes a protein shown as SEQ ID No.39 in a sequence table;

the sucA gene codes a protein shown by SEQ ID No.41 in a sequence table;

the protein shown by SEQ ID No.29 in the sequence table of the alkL gene coding sequence;

the protein shown by SEQ ID No.48 in the sequence table of the aspC gene coding sequence;

the panD gene encodes a protein shown by SEQ ID No.52 in a sequence table.

4. A method according to claim 2 or 3, characterized in that: the Escherichia coli is Escherichia coli BW 25113.

5. The method of claim 1, wherein:

a6 is realized by introducing the alkL gene into the recipient bacterium;

a7 is realized by introducing the gene in the L-aspartic acid synthesis pathway into the recipient bacterium;

a8 was achieved by introducing the panD gene into the recipient bacterium.

6. The method of claim 2, wherein: the fadR gene is a cDNA molecule or DNA molecule shown by SEQ ID No.1 in a sequence table;

the fabF gene is a cDNA molecule or DNA molecule shown by SEQ ID No.13 in a sequence table;

the fabH gene is a cDNA molecule or DNA molecule shown by SEQ ID No.15 in a sequence table;

the iclR gene is a cDNA molecule or a DNA molecule shown as SEQ ID No.38 in a sequence table;

the sucA gene is a cDNA molecule or DNA molecule shown by SEQ ID No.40 in a sequence table;

the alkL gene is a cDNA molecule or DNA molecule shown in SEQ ID No.28 in a sequence table;

the aspC gene is a cDNA molecule or DNA molecule shown by SEQ ID No.47 in a sequence table;

the panD gene is a cDNA molecule or DNA molecule shown by SEQ ID No.51 in a sequence table.

7. The method of claim 1, wherein:

the gdh gene is derived from Escherichia coli (Escherichia coli) or/and Bacillus subtilis (Bacillus subtilis);

the gene in the short chain fatty acid degradation regulation gene cluster atoSC gene cluster is a gene atoC gene for coding an atoC transcription activator and/or a gene atoS gene for coding an atoS-induced histidine kinase;

the gene in the short chain fatty acid degradation gene cluster atoDAEB gene cluster is a gene atoA gene for coding an acetoacetyl-CoA transferase alpha subunit, a gene atoD gene for coding an acetoacetyl-CoA transferase beta subunit, a gene atoE gene for coding an acetoacetate transporter and/or a gene atoB gene for coding acetyl-CoA acetyltransferase;

the glyoxylate pathway aceBA gene cluster comprises the following genes: the gene aceA coding for isocitrate lyase and the gene aceB coding for malate synthase.

8. The method of claim 1, wherein:

the gdh gene codes a protein shown by SEQ ID No.50 in a sequence table;

the protein shown by SEQ ID No.6 in the fadL gene coding sequence table;

the fadD gene codes a protein shown by SEQ ID No.9 in a sequence table;

the sthA gene encodes a protein shown by SEQ ID No.12 in a sequence table;

the atoSC gene cluster encodes the following proteins of a251) or a 261):

a251) protein shown by SEQ ID No.19 in a sequence table;

a261) protein shown as SEQ ID No.21 in a sequence table;

the aceBA gene cluster encodes the following proteins a271) and a 281):

a271) protein shown as SEQ ID No.44 in the sequence table;

a281) protein shown as SEQ ID No.46 in the sequence table.

9. The method according to claim 7 or 8, characterized in that:

b1 is achieved by introducing the gdh gene into the recipient bacterium;

b2 is prepared by replacing the promoter of said fadL gene with promoter P_CPA1The implementation is carried out;

b3 is a gene encoding a fatty acid beta-oxidation pathway, wherein the promoter P is a gene encoding a fatty acid beta-oxidation pathway_CPA1The implementation is carried out;

b4 is a gene obtained by replacing the promoter of the sthA gene with the promoter P_CPA1The implementation is carried out;

b5 is a gene product obtained by replacing the promoter of a gene in the short-chain fatty acid degradation pathway with the promoter P_CPA1The implementation is carried out;

b6 is prepared by replacing the promoter of the aceBA gene cluster with the promoter P_CPA1And (4) realizing.

10. The method of claim 9, wherein:

the promoter P_CPA1The coding sequence is DNA molecule at 1443-1622 of SEQ ID No.3 of the sequence table.

11. A method for producing beta-alanine, comprising: beta-alanine is prepared by biotransformation of the recombinant bacterium prepared by the method of any one of claims 1 to 10 using fatty acid as a substrate.

12. The method of claim 11, wherein: the fatty acid is palmitic acid, stearic acid, myristic acid, lauric acid, capric acid, caprylic acid and/or caproic acid.

13. A recombinant bacterium produced by the method of any one of claims 1 to 10.

14. The recombinant bacterium of claim 13, wherein any one of the following uses:

x1, producing beta-alanine;

x2, preparing and producing a beta-alanine product;

x3, degraded fatty acid;

and X4, and preparing a degraded fatty acid product.