CN118048380A - Corynebacterium glutamicum strain for producing 3-hydroxy propionic acid and construction method thereof - Google Patents

Abstract

The invention discloses a corynebacterium glutamicum strain for producing 3-hydroxy propionic acid and a construction method, comprising the following steps: (1) Introducing a maeB gene into a recombinant strain CY1 of corynebacterium glutamicum; replacing the promoter of the aceA gene with the P _sod* promoter; replacing the promoter of the aceB gene with the P _tuf promoter; knocking out rneG genes; replacement of the promoter of the isocitrate dehydrogenase icd gene with a growth-dependent promoter GPP 1; integrating two copies of mcr genes on a chromosome to obtain a strain CY8; (2) The pEC-sod-N-HP-C plasmid was introduced into CY8 to give a strain of corynebacterium glutamicum producing 3-hydroxypropionic acid. The strain constructed by the invention is safe and harmless, 3-hydroxy propionic acid is produced by taking acetic acid as a substrate through a simple fermentation process under the aerobic condition, 5.41g/L of 3-hydroxy propionic acid is produced in 60 hours, and the yield is 0.58g/g of acetic acid.

Description

Corynebacterium glutamicum strain for producing 3-hydroxy propionic acid and construction method thereof

Technical Field

The invention belongs to the field of bioengineering technology and application, and particularly relates to a corynebacterium glutamicum strain for producing 3-hydroxy propionic acid and a construction method thereof.

Background

3-Hydroxypropionic acid (3-hydroxypropionic acid, 3-HP) is a high value-added chemical that is an isomer with lactic acid (2-hydroxypropionic acid); in addition, hydroxyl groups and carboxyl groups at two ends of the 3-hydroxy propionic acid molecule endow 3-hydroxy propionic acid with active chemical properties, which means that the 3-hydroxy propionic acid molecule can be converted into other chemical substances such as acrylic acid, 1, 3-propanediol, poly (3-hydroxy propionate) and the like through different chemical reactions, and has wide application in the fields of medicine industry, agriculture, new materials and the like, and can be used for manufacturing medicine packaging materials, and also can be used for producing degradable plastics as substitutes ^[1] of common plastics. Based on the market value and application prospects of 3-hydroxypropionic acid, U.S. department of energy, 2004 and 2010, classified 3-hydroxypropionic acid twice as one of the 12 most promising and preferentially developed compounds ^[2]. The synthetic modes of 3-hydroxy propionic acid are divided into two types of chemical synthesis methods and biological synthesis methods. In recent years, with exhaustion of fossil fuels, the use of more sustainable microbial fermentation methods instead of chemical synthesis methods to produce 3-hydroxypropionic acid has attracted attention from researchers. The most widely studied routes for the synthesis of 3-hydroxypropionic acid using metabolic engineering pathways are the glycerol pathway, the beta-alanine pathway and the malonyl-coa pathway. Whereas in the malonyl-CoA pathway the standard Gibbs free energy from acetyl-CoA to 3-hydroxypropionic acid becomes-14.5 kJ/mol, it is thermodynamically favored ^[3]. The metabolites involved in this pathway are mostly intracellular common metabolites, and therefore, the malonyl-coa pathway can utilize a variety of substrates to produce 3-hydroxypropionic acid.

Various carbon sources are currently developed to synthesize 3-hydroxypropionic acid through various metabolic pathways, and the potential raw materials are glycerol, glucose, xylose, fatty acids, 1, 3-propanediol, acetic acid and the like. Acetic acid, a widely occurring monoacid, can be produced from lignocellulose and C1 gas, and is also a major byproduct of microbial fermentation. Acetic acid currently has a market price of $350-450 per ton, which is lower than $500 per ton of glucose; acetic acid has a market size of 1630 ten thousand tons in 2020 and is expected to reach 1960 ten thousand tons ^[4] by 2027. In addition, acetic acid can be utilized by a variety of microorganisms, and thus, the production of chemicals using acetic acid as a substrate has an economically viable advantage. In recent years, synthesis of various chemical substances, such as succinic acid, itaconic acid, mevalonic acid, and the like, has been studied using acetic acid as a single carbon source. Meanwhile, the acetyl-CoA is taken as an important precursor of the malonyl-CoA pathway, and the reaction pathway for generating the acetyl-CoA from acetic acid only needs two steps or one step, thereby being more beneficial to the production of 3-hydroxy propionic acid.

Corynebacterium glutamicum (Corynebacterium glutamicum) is an important industrial microorganism, has the advantages of clear genetic background, no pathogenicity, no sporulation, good food safety, and the like, and is a strain of GRAS authentication (GENERALLY RECOGNIZED AS SAFE), and is considered to be a very promising 3-hydroxypropionic acid production strain. Corynebacterium glutamicum can grow using acetic acid as the sole carbon source, and its intracellular mechanisms of transport, utilization and regulation of acetic acid have been thoroughly studied. Therefore, the production of 3-hydroxy propionic acid by using acetic acid as a single substrate in corynebacterium glutamicum has great application prospect. The current research on the production of chemicals using acetic acid as a substrate is mostly carried out in E.coli. Noh ^[5] et al, modified E.coli to produce 3.57g/L itaconic acid using acetic acid; xu ^[6] et al produced 7.85g/L mevalonic acid in recombinant E.coli using acetic acid as substrate by two-stage fermentation; lai ^[7] et al produced 15.8g/L of 3-hydroxypropionic acid in E.coli using whole cell catalysis with acetic acid and CO ₂ as substrates. These studies have all demonstrated the feasibility of producing various chemicals using acetic acid as a substrate, but these studies have been performed in E.coli. Compared with escherichia coli, corynebacterium glutamicum has better acid resistance, so that development of a corynebacterium glutamicum strain capable of utilizing acetic acid to produce 3-hydroxypropionic acid at high yield has important research significance. Chang ^[8] et al produced 4.26 g/L3-hydroxypropionic acid in shake flasks with acetic acid as the sole substrate and with simultaneous addition of cerulomycin to yield 0.50g/g acetic acid; 17.1 g/L3-hydroxypropionic acid can be produced in a 5L fermenter. After a series of metabolic engineering transformation and fermentation condition optimization, 30.5g/L of 3-hydroxypropionic acid is obtained in a 5L fermentation tank, and the yield is 0.17g/g of acetic acid (refer to the authorized patent (patent number: CN202210673552.3, corynebacterium glutamicum strain with high yield of 3-hydroxypropionic acid, construction method and application)). However, the shake flask yield and yield of the final engineering strain are still not ideal, so there is a need to develop Corynebacterium glutamicum strains which can utilize acetic acid and can obtain a higher yield of 3-hydroxypropionic acid.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a corynebacterium glutamicum strain for producing 3-hydroxy propionic acid.

A second object of the present invention is to provide a method for constructing a strain of Corynebacterium glutamicum which produces 3-hydroxypropionic acid.

It is a third object of the present invention to provide the use of the above-described strain of Corynebacterium glutamicum producing 3-hydroxypropionic acid in the production of 3-hydroxypropionic acid.

The technical scheme of the invention is summarized as follows:

A method for constructing a strain of corynebacterium glutamicum producing 3-hydroxypropionic acid, comprising the steps of:

(1) Introducing a maeB gene derived from escherichia coli into a corynebacterium glutamicum recombinant strain CY 1; replacing the promoter of the isocitrate lyase aceA gene with the P _sod* promoter; replacing the promoter of the malate synthase aceB gene with the P _tuf promoter; knocking out rneG genes encoding RNase E/G; replacement of the promoter of the isocitrate dehydrogenase icd gene with a growth-dependent promoter GPP 1; integrating two copies of mcr genes on a chromosome to obtain a strain CY8;

(2) Introducing pEC-sod-N-HP-C plasmid into the strain CY8 obtained in the step (1) to obtain a corynebacterium glutamicum strain for producing 3-hydroxy propionic acid, namely the strain CY9.

The nucleotide sequence of the maeB gene is shown as SEQ ID NO. 1;

the nucleotide sequence of the P _sod* promoter is shown as SEQ ID NO. 2;

The nucleotide sequence of the P _tuf promoter is shown as SEQ ID NO. 3;

the nucleotide sequence of rneG gene is shown in SEQ ID NO. 6;

The nucleotide sequence of the growth-dependent promoter GPP1 is shown as SEQ ID NO. 7;

The nucleotide sequence of the mcr gene is shown as SEQ ID NO. 9;

The corynebacterium glutamicum strain which is constructed by the method and used for producing 3-hydroxy propionic acid.

The application of the strain in the production of 3-hydroxy propionic acid.

The invention has the advantages that: the corynebacterium glutamicum strain for producing 3-hydroxy propionic acid constructed by the invention is safe and harmless, and uses acetic acid as a substrate to produce 3-hydroxy propionic acid through a simple fermentation process under an aerobic condition, 5.41g/L of 3-hydroxy propionic acid is produced in 60 hours, and the yield is 0.58g/g of acetic acid, which lays a foundation for subsequent industrialized production of 3-hydroxy propionic acid.

Drawings

FIG. 1 is a map of the vector pD-butA (in) -P _sod* -maeB plasmid with the heterologous maeB gene introduced at position butA.

FIG. 2 is a map of the vector pD-P _sod*-aceA-P_tuf -aceB plasmid with the aceA and aceB gene promoters replaced with the P _sod* and P _tuf promoters, respectively.

FIG. 3 is a map of rneG gene knockout vector pD-rneG.

FIG. 4 is a map of the vector pD-GPP1-icd plasmid with the icd gene promoter replaced with the growth-dependent promoter GPP 1.

FIG. 5 is a map of the vector pD-CgLP (in) -mcr-N-HP-C plasmid incorporating a copy of the mcr gene at chromosome CgLP's 4 locus.

FIG. 6 is a map of a vector pD-ldhA (in) -mcr-HP-C plasmid incorporating a second copy of the mcr-HP-C gene at the chromosomal ldhA site.

FIG. 7 is a map of the vector pD-ldhA (in) -mcr-N plasmid integrating the second copy of the mcr-N gene at the chromosomal ldhA site.

FIG. 8 is a fermentation production curve of Corynebacterium glutamicum CY9 producing 3-hydroxypropionic acid in 250ml shake flasks.

Detailed Description

The present invention is further illustrated with reference to the following examples, which are intended to enable a person skilled in the art to better understand the present invention, but are not intended to be limiting in any way.

The original strain Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 used in the present invention was derived from ATCC (AMERICAN TYPE culture collection, https:// www.atcc.org /), purchased 10 in 2012.

The recombinant strain CY1 of the corynebacterium glutamicum serving as an initial strain used in the invention is also called CW4, CW4 and plasmid pEC-sod-N-HP-C are obtained by construction with reference to the issued patent (patent number: CN202210673552.3, corynebacterium glutamicum strain with high yield of 3-hydroxy propionic acid, construction method and application).

Recombinant plasmid pK18mobsacB and expression vector pEC-XK99E were purchased from BioVector NTCC (http:// www.biovector.net /).

The 3-hydroxypropionic acid standard used was purchased from TCI company (https:// www.tcichemicals.com).

The molecular biological reagents used were purchased from Thermo (http:// www.thermoscientificbio.com/fermentas), and the seamless cloning kit was purchased from Beijing full gold (https:// www.transgen.com /).

Other biochemical reagents used were purchased from the company division of biological engineering (Shanghai) (http:// www.sangon.com /).

Example 1: introduction of the E.coli-derived Male B Gene into Corynebacterium glutamicum recombinant Strain CY1

The specific operation method and the used primers of the traceless operation technology of the corynebacterium glutamicum and the construction of a tool carrier pD-sacB (commodity) can refer to the corynebacterium glutamicum strain with the application number of CN201710215459.7 and high-yield chiral D- (-) -acetoin and the construction and application of the corynebacterium glutamicum strain;

Promoter P _sod(C131T) used in the present invention has been published ^[9] in the literature (the present invention names it as P _sod*); the integrative plasmid used, pD-sacB-butA, has been published in the literature (this invention names it pD-butA (in)) ^[10].

The specific procedure for introducing the E.coli-derived malic enzyme maeB gene (SEQ ID NO. 1) into recombinant strain CY1 of Corynebacterium glutamicum is as follows:

PCR amplification was performed with primers maeB-1 (SEQ ID NO. 21) and maeB-2 (SEQ ID NO. 22) using the laboratory-stored fragment of the P _sod* promoter (SEQ ID NO. 2) as template to obtain a fragment of the P _sod* promoter (SEQ ID NO. 2);

Carrying out PCR amplification by using ESCHERICHIA COLI K-12MG1655 (commodity) genome as a template and primers maeB-3 (SEQ ID NO. 23) and maeB-4 (SEQ ID NO. 24) to obtain maeB gene (SEQ ID NO. 1);

PCR amplification was performed with primers maeB-5 (SEQ ID NO. 25) and maeB-6 (SEQ ID NO. 26) using the laboratory-maintained integrative plasmid pD-butA (in) (SEQ ID NO. 10) as template to obtain linearized vector fragment pD-butA;

Purifying and recovering the fragments, fusing the P _sod* promoter fragment and the maeB fragment by using primers maeB-1 (SEQ ID NO. 21) and maeB-4 (SEQ ID NO. 24), and cutting and recovering to obtain a fused fragment P _sod* -maeB;

The fragment P _sod* -maeB and the linearization vector fragment pD-butA were subjected to seamless cloning, and transformed into E.coli DH 5. Alpha. After ligation, the butA locus introduced maeB gene integration vector pD-butA (in) -P _sod* -maeB was obtained (see FIG. 1). The plasmid with correct sequencing result is introduced into corynebacterium glutamicum CY1 through electrotransformation, and a butA locus is obtained through traceless operation technology to introduce a copy of maeB gene strain CY2.

Example 2: replacing the promoter of the isocitrate lyase aceA gene with the P _sod* promoter; replacement of the promoter of the malate synthase aceB Gene with the P _tuf promoter

PCR amplification is carried out by using a P _sod* promoter (SEQ ID NO. 2) fragment stored in a laboratory as a template and using primers sod-1 (SEQ ID NO. 27) and sod-2 (SEQ ID NO. 28) to obtain a P _sod* promoter fragment;

PCR amplification is carried out by using the genome of C.glutamicum ATCC 13032 as a template and using primers aceB-1 (SEQ ID NO. 29) and aceB-2 (SEQ ID NO. 30) to obtain an upstream homology arm aceB-U (SEQ ID NO. 11) of an aceB gene promoter;

PCR amplification with primers tuf-1 (SEQ ID NO. 31) and tuf-2 (SEQ ID NO. 32) gave a fragment of the P _tuf promoter (SEQ ID NO. 3);

The primers aceA-1 (SEQ ID NO. 33) and aceA-2 (SEQ ID NO. 34) were used for PCR amplification to obtain the homologous arm aceA-U (SEQ ID NO. 12) upstream of the aceA gene promoter.

After the above fragments were purified and recovered, the P _tuf promoter fragment and aceB-U fragment were fused with primers aceB-1 (SEQ ID NO. 29) and tuf-2 (SEQ ID NO. 32); fusing the P _sod* promoter fragment and the aceA-U fragment by using primers sod-1 (SEQ ID NO. 27) and aceA-2 (SEQ ID NO. 34), and recovering the gel to obtain fragment P _tuf-aceB-U、P_sod* -aceA-U;

And then fusing the P _tuf -aceB-U and the P _sod* -aceA-U fragments by using primers aceB-1 (SEQ ID NO. 29) and aceA-2 (SEQ ID NO. 34), and recovering the glue to obtain a fused fragment P _tuf-aceB-P_sod* -aceA.

The fusion products P _tuf-aceB-P_sod* -aceA and pD-sacB (commercial product) were digested with Thermo FAST DIGEST bamHI/XbaI, ligated and transformed into E.coli DH 5. Alpha. To give aceB and aceA gene promoters replacing vector pD-P _sod*-aceA-P_tuf -aceB (see FIG. 2). The plasmid with correct sequencing result is led into corynebacterium glutamicum CY2 through electrotransformation, and aceA and aceB gene promoters are obtained to replace strain CY3 through traceless operation technology.

Example 3: rneG Gene coding RNase E/G was knocked out

The specific operational procedure for knocking out rneG gene (SEQ ID NO. 6) encoding RNase E/G is as follows:

PCR amplification was performed using the genome of C.glutamicum ATCC 13032 as a template, with primers rneG-1 (SEQ ID NO. 35) and rneG-2 (SEQ ID NO. 36) to obtain the upstream homology arm rneG-U (SEQ ID NO. 13) of the rneG gene; PCR amplification with primers rneG-3 (SEQ ID NO. 37) and rneG-4 (SEQ ID NO. 38) gave the homology arm rneG-D (SEQ ID NO. 14) downstream of the rneG gene;

The linearized vector fragment pD-sacB-rneG was obtained by PCR amplification using the tool vector pD-sacB as a template and primers rneG-5 (SEQ ID NO. 39) and rneG-6 (SEQ ID NO. 40).

The fragments were purified and recovered, and the rneG-U and rneG-D fragments were fused using primers rneG-1 (SEQ ID NO. 35) and rneG-4 (SEQ ID NO. 38), and recovered by gel to give fusion fragment rneG-UD. The fragment rneG-UD and the linearization vector fragment pD-sacB-rneG were subjected to seamless cloning, and the pD-rneG plasmid was obtained after ligation and transformation (see FIG. 3). The plasmid with correct sequencing result is introduced into corynebacterium glutamicum CY3 through electrotransformation, and rneG gene knockout strain CY4 is obtained through traceless operation technology.

Example 4: replacement of the promoter of the isocitrate dehydrogenase gene icd with the growth-dependent promoter GPP1

The growth-dependent promoter GPP1 used in the present invention has been reported ^[11] in the literature.

PCR amplification was performed with primers GPP1-1 (SEQ ID NO. 41) and GPP1-2 (SEQ ID NO. 42) using the genome of C.glutamicum ATCC 13032 as a template to obtain a growth-dependent promoter GPP1 (SEQ ID NO. 7) fragment; PCR amplification with primers icd-1 (SEQ ID NO. 43) and icd-2 (SEQ ID NO. 44) to obtain icd gene upstream homology arm icd-U (SEQ ID NO. 15); the primers icd-3 (SEQ ID NO. 45) and icd-4 (SEQ ID NO. 46) were used for PCR amplification to obtain the homologous arm icd-D (SEQ ID NO. 16) downstream of the icd gene. The above-mentioned fragments are purified and recovered,

GPP1, icd-U and icd-D fragments were fused with primers icd-1 (SEQ ID NO. 43) and icd-4 (SEQ ID NO. 46) to give fusion products icd-U-GPP1-D.

The fusion products icd-U-GPP1-D and pD-sacB were double digested with Thermo FAST DIGEST bamHI/salI, ligated and transformed into E.coli DH 5. Alpha. To give the icd gene promoter replacement vector pD-GPP1-icd (see FIG. 4). The plasmid with correct sequencing result is introduced into corynebacterium glutamicum CY4 through electrotransformation, and the icd gene promoter substitution strain CY5 is obtained through a traceless operation technology.

Example 5: insertion of a copy of malonyl-CoA reductase Gene mcr at position CgLP on chromosome

The CgLP th site used in the present invention has been reported ^[12] in the literature.

PCR amplification was performed using the genome of C.glutamicum ATCC 13032 as a template, with primers CgLP4-1 (SEQ ID NO. 47) and CgLP-2 (SEQ ID NO. 48) to give a homology arm CgLP4-U (SEQ ID NO. 17) upstream of the CgLP locus; PCR amplification with primers CgLP4-3 (SEQ ID NO. 49) and CgLP-4 (SEQ ID NO. 50) gave homology arm CgLP-D (SEQ ID NO. 18) downstream of the CgLP locus;

PCR amplification is carried out by using plasmid pEC-sod-N-HP-C as a template and using a primer mcr-1 (SEQ ID NO. 51) and a primer mcr-2 (SEQ ID NO. 52) to obtain an mcr gene (SEQ ID NO. 9) fragment; PCR amplification was performed using primers rrnB-1 (SEQ ID NO. 53) and rrnB-2 (SEQ ID NO. 54) to obtain terminator rrnB (SEQ ID NO. 19) fragment;

The linearized vector fragment pD-sacB-mcr was obtained by PCR amplification using the tool vector pD-sacB as a template and the primers mcr-3 (SEQ ID NO. 55) and mcr-4 (SEQ ID NO. 56). The above fragments were purified and recovered, and CgLP-U and mcr fragments were fused using primers CgLP-4-1 (SEQ ID NO. 47) and mcr-2 (SEQ ID NO. 52); the rrnB and CgLP-D fragments are fused by using primers rrnB-1 (SEQ ID NO. 53) and CgLP-4 (SEQ ID NO. 50), and the fusion fragment CgLP-U-mcr, rrnB-CgLP-D is obtained after glue recovery; the CgLP-U-mcr and rrnB-CgLP-D fragments were fused with primers CgLP-1 (SEQ ID NO. 47) and CgLP-4 (SEQ ID NO. 50), and recovered with gel to give fusion fragment CgLP-U-mcr-rrnB-D.

The fusion fragment CgLP-U-mcr-rrnB-D and the linearization vector fragment pD-sacB-mcr are subjected to seamless cloning, and pD-CgLP (in) -mcr-N-HP-C plasmid is obtained after connection and transformation (see figure 5). The plasmid with correct sequencing result is led into corynebacterium glutamicum CY5 through electrotransformation, and bacterial strain CY6 with CgLP site inserted into one copy mcr gene on chromosome is obtained through traceless operation technology.

Example 6: insertion of a second copy of malonyl-CoA reductase Gene at the C-terminal mcr-C in the ldhA site on the chromosome

The integrative plasmid pD-sacB-ldhA used in the present invention has been published in the literature (this invention designates it as pD-ldhA (in)) [10].

The mcr-N, mcr-C gene and the first 62bp fragment of clpP gene used in the invention refer to the issued patent (patent number: CN202210673552.3, corynebacterium glutamicum strain with high yield of 3-hydroxy propionic acid, construction method and application).

PCR amplification was performed with primers sod-mcr-1 (SEQ ID NO. 57) and sod-mcr-2 (SEQ ID NO. 58) using plasmid pEC-sod-N-HP-C as template to obtain the P _sod* fragment; PCR amplification is carried out by using a primer mcr-C-1 (SEQ ID NO. 59) and an mcr-C-2 (SEQ ID NO. 60) to obtain an mcr-C fragment with a pre-62 bp fragment of the clpP gene; PCR amplification was performed using the laboratory-maintained integrative plasmid pD-ldhA (in) (SEQ ID NO. 20) as a template and primers ldhA-1 (SEQ ID NO. 61) and ldhA-2 (SEQ ID NO. 62) to obtain linearized vector fragment pD-ldhA; purifying and recovering the fragments, fusing the P _sod* -mcr and the mcr-C fragments by using primers sod-mcr-1 (SEQ ID NO. 57) and mcr-C-2 (SEQ ID NO. 60), and cutting and recovering to obtain fused fragments P _sod* -mcr-HP-C; the fragment P _sod* -mcr-HP-C and the linearized vector fragment pD-ldhA were seamlessly cloned, ligated and transformed into E.coli DH 5. Alpha. To obtain a second copy of the mcr-C integration vector pD-ldhA (in) -mcr-HP-C inserted on the chromosome (see FIG. 6). The plasmid with correct sequencing result is introduced into corynebacterium glutamicum CY6 through electrotransformation, and the second copy mcr-HP-C gene strain CY7 inserted into the ldhA locus on the chromosome is obtained through a traceless operation technology.

Example 7: insertion of a second copy of the malonyl-CoA reductase Gene at the N-terminal mcr-N in the ldhA site on the chromosome

PCR amplification was performed with the primers mcr-N-1 (SEQ ID NO. 63) and mcr-N-2 (SEQ ID NO. 4) using plasmid pEC-sod-N-HP-C as template to obtain the mcr-N fragment; PCR amplification was performed using the laboratory-maintained integrative plasmid pD-ldhA (in) as a template and the primers mcr-N-3 (SEQ ID NO. 5) and mcr-N-4 (SEQ ID NO. 8) to obtain the linearized vector fragment pD-ldhA-U. After purification and recovery, the fragment mcr-N and the linearized vector fragment pD-ldhA-U were subjected to seamless cloning, and after ligation, transformed into E.coli DH 5. Alpha. To obtain a chromosome into which a second copy of the mcr-N integrating vector pD-ldhA (in) -mcr-N was inserted (see FIG. 7). The plasmid with correct sequencing result is introduced into corynebacterium glutamicum CY7 through electrotransformation, and the second copy mcr-N gene strain CY8 inserted into the ldhA locus on the chromosome is obtained through a traceless operation technology.

Example 8: shake flask fermentation of corynebacterium glutamicum strains producing 3-hydroxypropionic acid

The pEC-sod-N-HP-C plasmid was introduced into Corynebacterium glutamicum CY8 by electrotransformation, resulting in strain CY9 (Corynebacterium glutamicum strain producing 3-hydroxypropionic acid) which was non-inducible over-expressed with the free multicopy plasmid, the gene mcr of the 3-hydroxypropionic acid synthesis pathway

The strain codes such as CY1-CY9 and the like in the invention are for convenience of description, but are not to be construed as limiting the invention.

The inoculation mode is as follows: a proper amount of CY9 strain preserved at-80 ℃ is picked by a sterile inoculating loop, streaked on a BHI plate containing 25 mug/mL kanamycin, cultured for 48 hours in a 30 ℃ incubator, and single colony is picked to a test tube containing 5mL BHI containing 25 mug/mL kanamycin; culturing at 30deg.C in a shaker at 220rpm for about 12h, transferring to 50mL CGIII medium containing 25 μg/mL kanamycin and 20g/L glucose (inoculum size 2%); the culture was continued at 30℃for about 12 hours in a shaker at 220rpm, and the seed solution was transferred to a 250ml shake flask (initial OD ₆₀₀ of 0.5, initial acetic acid concentration of about 10 g/L) containing 50ml CGXII medium. After culturing at 30deg.C in a shaker at 220rpm for 72 hours, the fermentation was ended, and samples were taken every 12 hours to detect the OD ₆₀₀, acetic acid concentration and 3-HP concentration of the samples.

The BHI medium was: weighing 74g of brain heart infusion broth into a measuring cup, fixing volume to 1L with single distilled water, mixing uniformly, sterilizing at 121deg.C for 20min, and standing at normal temperature.

CGIII the formula of the culture medium is as follows: 3- (N-morpholino) propanesulfonic acid (21 g/L), yeast extract (10 g/L), tryptone (10 g/L), naCl (2.5 g/L), pH=7.0 adjusted with 5M NaOH solution, and sterilized at 121℃for 20min under 0.1 MPa.

The formula of the CGXII culture medium is as follows: 3- (N-morpholinium) propanesulfonic acid (21 g/L), ammonium sulfate (20 g/L), sodium acetate (14 g/L), yeast extract (10 g/L), urea (5 g/L), dipotassium hydrogen phosphate (1 g/L), potassium dihydrogen phosphate (1 g/L), magnesium heptahydrate (0.25 g/L), calcium chloride (10 mg/L), ferrous sulfate heptahydrate (10 mg/L), manganese sulfate monohydrate (10 mg/L), zinc sulfate heptahydrate (1 mg/L), copper sulfate (0.2 mg/L), nickel chloride monohydrate (20. Mu.g/L), biotin (0.2 mg/L), vitamin B1 (0.2 mg/L), and sterilization at 121℃for 20min under 0.1 MPa.

From the fermentation result, 5.41g/L of 3-hydroxy propionic acid is produced at 60 hours, and the yield is 0.58g/g of acetic acid (see figure 8), which lays a foundation for the subsequent industrialized production of 3-hydroxy propionic acid.

The corynebacterium glutamicum strain for producing 3-hydroxy propionic acid constructed by the invention can obtain the highest 3-hydroxy propionic acid yield and yield in a shake flask, and has good application prospect.

The genotype of the strain constructed by the invention is C.glutamicum ATCC 13032ΔldhΔmsmAΔcg0635ΔrneG-GPP1-icd,P_P1-gltA^ATG→TTG,P_P7-fasAΔfasO(accBC)ΔfasO(accD1),butA::P_sod*-maeB,P_tuf-aceB-P_sod*-aceA,CgLP4::P_sod*-N-HP-C*,ldhA::P_sod*-N-HP-C*;pEC-sod-N-HP-C*., and the strain can produce 5.41g/L of 3-hydroxypropionic acid by utilizing acetic acid.

The order of the steps of the construction of the strain of the present invention is not limited, and it is within the scope of the present invention for a person skilled in the art to achieve the object of the present invention according to the present disclosure.

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Claims (4)

1. A construction method of a corynebacterium glutamicum strain for producing 3-hydroxy propionic acid is characterized by comprising the following steps:

(1) Introducing a maeB gene derived from escherichia coli into a corynebacterium glutamicum recombinant strain CY 1; replacing the promoter of the isocitrate lyase aceA gene with the P _sod* promoter; replacing the promoter of the malate synthase aceB gene with the P _tuf promoter; knocking out rneG genes encoding RNase E/G; replacement of the promoter of the isocitrate dehydrogenase icd gene with a growth-dependent promoter GPP 1; integrating two copies of mcr genes on a chromosome to obtain a strain CY8;

(2) Introducing pEC-sod-N-HP-C plasmid into the strain CY8 obtained in the step (1) to obtain a corynebacterium glutamicum strain for producing 3-hydroxy propionic acid, namely the strain CY9.

2. The method according to claim 1, characterized in that said:

the nucleotide sequence of the maeB gene is shown as SEQ ID NO. 1;

the nucleotide sequence of the P _sod* promoter is shown as SEQ ID NO. 2;

The nucleotide sequence of the P _tuf promoter is shown as SEQ ID NO. 3;

the nucleotide sequence of rneG gene is shown in SEQ ID NO. 6;

The nucleotide sequence of the growth-dependent promoter GPP1 is shown as SEQ ID NO. 7;

The nucleotide sequence of the mcr gene is shown as SEQ ID NO. 9.

3. A strain of corynebacterium glutamicum constructed by the method of claim 1 or 2, which produces 3-hydroxypropionic acid.

4. Use of a strain according to claim 3 for the production of 3-hydroxypropionic acid.