CN114149955B

CN114149955B - Genetically engineered bacterium for efficiently producing glycerol and construction method and application thereof

Info

Publication number: CN114149955B
Application number: CN202111499125.XA
Authority: CN
Inventors: 马春玲; 王艺纯; 王瑞明; 李丕武; 苏静; 汪俊卿
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2023-09-26
Anticipated expiration: 2041-12-09
Also published as: CN114149955A

Abstract

The invention relates to a genetically engineered bacterium for efficiently producing glycerol, and a construction method and application thereof. According to the invention, the glycerol 3-phosphate dehydrogenase and the glycerol 3-phosphate enzyme after the optimized codons are enhanced in escherichia coli are constructed, so that when the engineering bacteria are used for producing glycerol by taking glucose as a substrate through fermentation, the glycerol yield is greatly improved, especially, the glycerol yield of recombinant escherichia coli E.coli Rosetta/pRSFduet-gpd1o-gpp2o in a 1L fermentation tank reaches 10.89g/L, and the glycerol yield in shake flask fermentation reaches 6.816g/L, compared with the shake flask fermentation of a control strain E.coli BL21 (de 3)/pRSFduet-gpd 1-gpp2 for expressing the non-optimized codons.

Description

Genetically engineered bacterium for efficiently producing glycerol and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a genetically engineered bacterium for efficiently producing glycerol, and a construction method and application thereof.

Background

Glycerol, also known as 1,2, 3-propanediol, is a colorless and odorless organic liquid, and is widely used in industries such as food, paper, cosmetics, leather, textile, photographic, printing, metal processing, electrical materials, rubber, and the like, as well as raw materials for producing various chemicals, in addition to being used as an antifreezing agent for automobile and aircraft fuels and oil fields. In addition, it is also considered as a raw material for industrial fermentation, and some products with high added value, such as xanthan gum, trehalose, rhamnolipid, citric acid, lactic acid, DHA, EPA,1, 3-propanediol, etc., are mainly produced by microbial fermentation, wherein 1,3-propanediol (1, 3-propanediol,1, 3-PD) is used as a fiber monomer of a novel polymer material, namely polytrimethylene terephthalate (PTT), which is one of more classical and advantageous products. The PTT fiber integrates the characteristics of softness, fluffiness of acrylic fibers, dirt resistance of terylene, inherent elasticity, normal-temperature dyeing and the like, integrates the excellent wearability of various fibers, and becomes one of the novel polymer materials newly developed internationally at present. About 55% of the PTT fibers are required from the carpet field, the remaining 45% being other textile fields, estimated by the expert concerned. However, the market price of the PTT is high at present, so that limited development results can be obtained only in the aspects of products and varieties with high cost digestion capacity, and the synthesis of PTT fiber monomer 1,3-propanediol by a microbial fermentation method becomes a hot spot for research of current domestic and foreign scholars.

In addition, the unique physical properties of 1,3-PD make it a good solvent, protective agent and anti-freezing agent, which have been recognized as GRAS (check mark used by the United states FDA) in 1998, and can be used as emulsifying agent, flavoring agent, thickening agent, antistaling agent, additive and moisture absorbent in the food field, thus not only improving the sensory quality of food, but also improving the storage resistance and stability of food, and simultaneously greatly improving the nutritional value of food.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a genetically engineered bacterium for efficiently producing glycerol, and a construction method and application thereof. According to the invention, an intermediate dihydroxyacetone phosphate is generated by utilizing substrate glucose through an EMP (electron emission protein) way in escherichia coli, and key enzymes 3-phosphoglycerate enzyme and 3-phosphoglycerate dehydrogenase which are obtained by optimizing two codons from saccharomyces cerevisiae are introduced and are over-expressed, so that the conversion of dihydroxyacetone phosphate into glycerol is realized, and a channel for producing glycerol by utilizing glucose is constructed.

The technical scheme of the invention is as follows:

a genetic engineering bacterium for efficiently producing glycerol, wherein the genetic engineering bacterium simultaneously strengthens and expresses 3-phosphoglycerate dehydrogenase and 3-phosphoglycerate enzyme after optimizing codons.

According to the invention, the genetically engineered bacterium is preferably recombinant escherichia coli, and the escherichia coli is E.coli BL21 (de 3), E.coli BL21 (star) or E.coli Rosetta.

According to the invention, the 3-phosphoglycerate dehydrogenase is a 3-phosphoglycerate dehydrogenase from saccharomyces cerevisiae, the coding gene of the 3-phosphoglycerate dehydrogenase is gpd1o, and the nucleotide sequence of the 3-phosphoglycerate dehydrogenase is shown as SEQ ID NO. 2. The codon of the enzyme is optimally designed, so that the enzyme is more suitable for escherichia coli.

According to the invention, the 3-phosphoglycerase is 3-phosphoglycerase from Saccharomyces cerevisiae, the coding gene of the 3-phosphoglycerase is gpp2o, and the nucleotide sequence of the 3-phosphoglycerase is shown as SEQ ID NO. 4. The codon of the enzyme is optimally designed, so that the enzyme is more suitable for escherichia coli.

According to the present invention, it is preferable that the expression vector used for constructing the genetically engineered bacterium has a glycerol-3-phosphate dehydrogenase gene located upstream of the glycerol-3-phosphate enzyme.

According to the invention, preferably, when the genetically engineered bacterium is recombinant E.coli BL21 (de 3), chaperones 1 and 2 are inserted into the genetically engineered bacterium.

Further preferably, the molecular chaperone 1 is derived from escherichia coli, the encoding gene of the molecular chaperone 1 is gril, and the nucleotide sequence of the molecular chaperone 1 is shown as SEQ ID NO. 5; the molecular chaperone 2 is derived from escherichia coli, the coding gene of the molecular chaperone 2 is gros, and the nucleotide sequence of the molecular chaperone 2 is shown as SEQ ID NO. 6.

Further preferably, the molecular chaperone 1 in the expression vector used for constructing the genetically engineered bacterium is positioned at the downstream of the 3-phosphoglycerate dehydrogenase and at the upstream of the 3-phosphoglycerate dehydrogenase; chaperone 2 is located downstream of 3-phosphoglycerase.

The genetically engineered bacterium of the invention is adopted for shake flask fermentation for 70 hours, and the glycerol yield is improved by 58 times compared with a control strain.

The construction method of the genetically engineered bacterium comprises the following steps:

(1) Inserting a glycerol 3-phosphate dehydrogenase gene gpd1o of saccharomyces cerevisiae into a vector plasmid pRSFdure to construct an expression vector pRSFdure-gpd 1o;

(2) Inserting a 3-phosphoglycerase gene gpp2o of saccharomyces cerevisiae into a vector plasmid pRSFduet-gpd1o to construct an expression vector pRSFduet-gpd1o-gpp2o;

(3) Inserting a molecular chaperone 1 gene grol of escherichia coli into a vector plasmid pRSFduet-gpd1o-gpp2o to construct an expression vector pRSFduet-gpd1o-gpp2o-grol;

(4) Inserting a molecular chaperone 2 gene gros of escherichia coli into a vector plasmid pRSFduet-gpd1o-gpp2o-grol to construct an expression vector pRSFduet-gpd1o-gpp2o-grol-gros;

(5) The expression vector pRSFduet-gpd1o-gpp2o is respectively transformed into E.coli BL21 (star) and E.coli Rosetta, the expression vector pRSFduet-gpd1o-gpp2o-grol-gros is transformed into E.coli BL21 (de 3), positive recombinants are selected, and the genetic engineering bacteria E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros, E.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o and E.coli Rosetta/pRSFduet-gpd1o-gpp2o are obtained.

According to the preferred embodiment of the invention, the construction method of the genetically engineered bacterium specifically comprises the following steps:

(1) Performing PCR amplification by taking a Saccharomyces cerevisiae S288C genome as a template to obtain a gpd1o sequence of 3-phosphoglycerate dehydrogenase, wherein the PCR primer sequence is as follows:

primer1:5'-cgcctgcaggtcgacaagcttATGAGCGCGGCGGCTGAC-3' (containing BamHI cleavage site),

primer2:5'-agctgccatctccttgcggccgcTTAGTCCTCGTGCAGATCCAGTT-3' (HindIII cleavage site);

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 60 ℃ for 40sec; extension, 72 ℃ for 30sec (30 cycles); stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

after vector plasmid pRSFduet is digested with HindIII and NotI, pRSFduet-gpd1o plasmid is obtained by connecting a one-step cloning kit;

(2) Performing PCR amplification by taking a Saccharomyces cerevisiae S288C genome as a template to obtain a 3-phosphoglyceride gene gpp2o sequence, wherein the PCR primer sequence is as follows:

primer3:5'-taagaaggagatatacatatg ATGGGTCTGACCACCAAACCG-3' (containing Nde I cleavage site),

primer4:5'-gccgatatccaattgagatct TTACCATTTCAGCAGATCATCTTTCG-3' (Bgl II cleavage site);

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 60 ℃ for 30sec; extension, 72 ℃ for 90sec (30 cycles); stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet-gpd1o obtained in the step (1) is subjected to double digestion by Nde I and Bgl II respectively, and then is connected by a one-step cloning kit to obtain pRSFduet-gpd1o-gpp2o plasmid;

(3) Performing PCR amplification by taking the escherichia coli MG1655 genome as a template to obtain a molecular chaperone 1 gene grol sequence, wherein the PCR primer sequence is as follows:

primer5:5'-caggtcgacaagcttgcggccgc AAGGAGATGGCAGCTAAAGACGTAAAATTCG-3' (containing Not I cleavage site),

primer6:5'-ttactttctgttcgacttaag TTACATCATGCCGCCCATG-3' (containing Afl II cleavage site);

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 65 ℃ for 30sec; extension, 45s (30 cycles) at 72 ℃; stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet-gpd1o-gpp2o obtained in the step (2) is subjected to double digestion by using Not I and Afl II, and then is connected by a one-step cloning kit to obtain pRSFduet-gpd1o-gpp2o-grol plasmid;

(4) Performing PCR amplification by taking the escherichia coli MG1655 genome as a template to obtain a molecular chaperone 2 gene gros sequence, wherein the PCR primer sequence is as follows:

primer7:5'-gtctactagcgcagcttaattaaAAGGAGATGAATATTCGTCCATTGCATGAT-3' (containing Pac I cleavage site),

primer8:5'-cagcggtggcagcagcctaggTTACGCTTCAACAATTGCCAGA-3' (containing Avr II cleavage site);

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 65 ℃ for 30sec; extension, 15s (30 cycles) at 72 ℃; stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet-gpd1o-gpp2o-grol obtained in the step (3) is subjected to double digestion by Pac I and Avr II, and then is connected by a one-step cloning kit to obtain pRSFduet-gpd1o-gpp2o-grol-gros plasmid;

(5) And (3) chemically transforming pRSFduet-gpd1o-gpp2o plasmids obtained in the step (2) into escherichia coli competent cells BL21 (star) and Rosetta respectively, chemically transforming pRSFduet-gpd1o-gpp2o-grol-gros plasmids obtained in the step (4) into escherichia coli competent cells BL21 (de 3), and selecting positive recombinants to obtain genetically engineered bacteria E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros, E.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o and E.coli Rosetta/pRSFduet-gpd1o-gpp2o.

The application of the genetically engineered bacterium in the production of glycerol.

According to the invention, the application is to ferment and produce glycerol by taking glucose as a substrate, and the production process is as follows:

activating the genetically engineered bacteria on an LB solid culture medium, picking an activated single colony, inoculating the single colony into an LB liquid culture medium, culturing for 12 hours at 37 ℃ and 200r/min, inoculating the single colony into a fermentation culture medium according to the inoculum size of 2% -10%, and fermenting at 37 ℃ to produce glycerol;

wherein, the fermentation medium composition (/ L): mgSO (MgSO) ₄ ·7H ₂ O 0.48g，NaH ₂ PO ₄ 6.0g，CaCl ₂ 0.0111g，NH ₄ Cl2.0g，NaCl 0.5g，KH ₂ PO ₄ 3.0g, yeast extract powder 5.0g and glucose 40g.

The invention has the beneficial effects that:

1. according to the invention, the 3-phosphoglycerate dehydrogenase and the 3-phosphoglycerate enzyme with optimized codons are simultaneously expressed in escherichia coli, and a new genetic engineering bacterium is constructed, and because two key enzyme genes from eukaryotic cells are optimized for codons, the novel genetic engineering bacterium is more suitable for expression in escherichia coli, when the engineering bacterium is used for producing glycerol by taking glucose as a substrate through fermentation, the glycerol yield is greatly improved, especially, the glycerol yield of recombinant escherichia coli E.coli Rosetta/pRSFduet-gpd1o-gpp2o in a 1L fermentation tank reaches 10.89g/L, and the glycerol yield in shake flask fermentation reaches 6.816g/L, compared with the shake flask fermentation of a control strain E.coli BL21 (de 3)/pRSFduet-gpd 1-gpp2 for expressing the 3-phosphoglycerate dehydrogenase with non-optimized codons, the glycerol yield is improved by 58 times.

2. The invention selects E.coli BL21 (de 3), E.coli BL21 (star) or E.coli Rosetta as host bacteria when constructing genetic engineering bacteria, has higher glycerol tolerance and conversion rate, belongs to facultative bacteria, and can ferment under micro-aerobic or anaerobic conditions. The metabolic process of the escherichia coli is clear, the operation is simple and convenient, convenience is brought to the genetic improvement of the strain and the construction of a new strain by utilizing genetic engineering, in addition, the stability of mRNA in a host cell can be enhanced by the host strain E.coli BL21 (star), the expression level of exogenous genes, especially eukaryotic genes, can be further improved, the host strain E.coli Rosetta carries chloramphenicol resistance, and the tRNA corresponding to six rare codons lacking in the escherichia coli is supplemented, so that the expression level of the eukaryotic genes in prokaryotic cells is further improved, the glycerol yield in shake flask fermentation reaches 5.318g/L and 6.816g/L respectively, and compared with shake flask fermentation of a recombinant strain E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o using E.coli BL21 (de 3) as host bacteria, the glycerol yield is improved by 15 times. When the host bacterium is E.coli BL21 (de 3), the insertion of the molecular chaperone 1 and the molecular chaperone 2 into the genetically engineered bacterium can further improve the yield of glycerin compared with a control strain, and the yield reaches 4.425/L in shake flask fermentation, which is 25 times that of the control strain E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o without the insertion of the molecular chaperone.

Drawings

FIG. 1 is a map of four plasmids; in the figure, A is pRSFduet-gpd1o plasmid, B is pRSFduet-gpd1o-gpp2o plasmid, C is pRSFduet-gpd1o-gpp2o-grol plasmid, and D is pRSFduet-gpd1o-gpp2o-grol-gros plasmid.

FIG. 2 is a colony PCR gel electrophoresis of positive recombinant E.coli/pRSFduet-gpd 1o;

in the figure, each lane is a gpd1o gene fragment band.

FIG. 3 is a colony PCR gel electrophoresis of positive recombinant E.coli/pRSFduet-gpd1o-gpp 2o;

in the figure, each lane is a gpp2o gene fragment band.

FIG. 4 is a colony PCR gel electrophoresis diagram of positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros;

lanes 1,2,3 are the gri gene fragment bands of E.coli BL21 (de 3)/pRSFdure-gpd 1o-gpp2 o-gri-gros colonies; lanes 4,5,6 are bands of gros gene fragments of E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros colonies.

FIG. 5 is an SDS-PAGE gel of positive recombinant E.coli/pRSFduet-gpd1o-gpp 2o;

in the figure, lane 1 represents E.coli Rosetta/pRSFDuet-gpd1o-gpp2o, lane 2 represents E.coli BL21 (star)/pRSFDuet-gpd 1o-gpp2o, lane 3 represents E.coli BL21 (de 3)/pRSFDuet-gpd 1o-gpp2o, and lanes 4,5 represent E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros.

FIG. 6 is a diagram of positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1-gpp2; glycerol yield comparison plot for 70h of coll BL21 (de 3)/pRSFduet-gpd 1o-gpp2o shake flask fermentation.

FIG. 7 is an additive chaperone positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o; e.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol; glycerol yield comparison plot of E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros shake flask fermentation for 70 h;

FIG. 8 is a diagram of E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o of different host cell positive recombination; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; glycerol yield comparison plot for 70h of coll Rosetta/pRSFduet-gpd1o-gpp2o shake flask fermentation.

FIG. 9 is a graph showing comparison of glycerol yields of positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o fermented in a 1L mini-fermenter for 70h.

FIG. 10 is a graph showing comparison of glycerol yields of positive recombinant E.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o fermented in a 1L mini-fermentor for 70h.

FIG. 11 is a graph showing comparison of glycerol yields of positive recombinant E.coli Rosetta/pRSFduet-gpd1o-gpp2o fermented in a 1L mini-fermentor for 70h.

Detailed description of the preferred embodiments

The technical scheme of the present invention will be further described with reference to examples and drawings, but the scope of the present invention is not limited thereto. The reagents and medicines related to the examples are common commercial products unless specified; the experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art.

The Saccharomyces cerevisiae and the escherichia coli used in the invention are common commercial strains, and can be purchased from microorganism collection centers or strain sales companies, and the one-step cloning kit and the plasmid extraction kit used in the invention are respectivelyOne Step Cloning Kit C112-01，/>Plasmid Mini Kit DC201-01, available from Nanjinovirginia Biotech Co., ltd.

Example 1: construction of plasmid pRSFduet-gpd1o

The coding gene of the 3-phosphoglycerate dehydrogenase in the Saccharomyces cerevisiae S288C is gpd1, the nucleotide sequence is shown as SEQ ID NO.1, and the codon is optimized according to the codon preference to obtain the optimized 3-phosphoglycerate dehydrogenase, the coding gene is named gpd1o, and the nucleotide sequence is shown as SEQ ID NO. 2. The amplification primers Primer1 and Primer2 of the gpd1o sequence are designed by using bioinformatics software, and then PCR amplification is carried out by taking the Saccharomyces cerevisiae S288C genome as a template, so as to obtain the gpd1o sequence.

The primer sequences were as follows:

the PCR amplification system is prepared according to the instruction of the kit;

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 60 ℃ for 30sec; extension, 72 ℃ for 30sec (30 cycles); stopping extension at 72 ℃ for 10min; finally, the temperature is kept at 4 ℃.

The PCR product is analyzed and detected by agarose gel electrophoresis with the concentration of 1%, an electrophoresis band with the size of about 1176bp is obtained, a target fragment is recovered by a gel recovery kit, a vector plasmid pRSFduet is subjected to double enzyme digestion by BamH I and Hind III, pRSFduet-gpd1o plasmid is obtained by one-step cloning enzyme connection, pRSFduet-gpd1o plasmid is transferred into competent cells of escherichia coli BL21 (de 3) by a chemical conversion method, and the competent cells are coated on a kanamycin solid LB plate containing 50 mug/mL for overnight culture at 37 ℃, and positive recombinants are selected for storage at the temperature of minus 80 ℃; the recombinant bacterium is named as E.coli BL21 (de 3)/pRSFduet-gpd 1o; pRSFduet-gpd1o plasmid was extracted from recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o using a plasmid extraction kit, and its plasmid map is shown in FIG. 1A.

Example 2: construction of plasmid pRSFduet-gpd1o-gpp2 o:

the coding gene of 3-phosphoglycerase in Saccharomyces cerevisiae S288C is gpp2, the nucleotide sequence is shown as SEQ ID NO.3, and the codon is optimized according to the codon preference to obtain the optimized 3-phosphoglycerase, the coding gene is named as gpp2o, and the nucleotide sequence is shown as SEQ ID NO. 4. The amplification primers Primer3 and Primer4 of the gpp2o sequence are designed by using bioinformatics software, and then PCR amplification is carried out by taking the Saccharomyces cerevisiae S288C genome as a template, so as to obtain the gpp2o sequence.

The primer sequences were as follows:

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 65 ℃ for 30sec; extension, 72 ℃ for 30sec (30 cycles); stopping extension at 72 ℃ for 10min; finally, the temperature is kept at 4 ℃.

The PCR product is detected and analyzed by agarose gel electrophoresis with the concentration of 1 percent to obtain an electrophoresis band with the size of about 700bp, and the target fragment is recovered by a gel recovery kit.

Cloning the optimized 3-phosphoglycerase gene gpp2o into pRSFduet-gpd1o plasmid, and constructing pRSFduet-gpd1o-gpp2o plasmid by positioning the optimized 3-phosphoglycerase gene gppp2o at the downstream of the promoter T7-promoter 2.

The method comprises the following specific steps: after vector plasmid pRSFduet-gpd1o is subjected to double digestion by Nde I and Bgl II, the pRSFduet-gpd1o-gpp2o plasmid is obtained by connecting a one-step cloning kit, pRSFduet-gpd1o-gpp2o plasmid is transferred into competent cells of escherichia coli BL21 (de 3) by a chemical transformation method, the competent cells are coated on a kanamycin solid LB plate containing 50 mu g/mL for overnight culture at 37 ℃, positive recombinants are picked up and stored at-80 ℃, and the recombined bacteria are named as E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o; the pRSFduet-gpd1o-gpp2o plasmid was extracted from recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o using a plasmid extraction kit, and its plasmid map is shown in FIG. 1B.

Example 3: construction of plasmid pRSFduet-gpd1o-gpp2o-grol

Cloning chaperone 1 gene grol in E.coli into pRSFduet-gpd1o-gpp2o plasmid, wherein the chaperone 1 gene grol is positioned downstream of the promoter T7-promoter1 gpd1o gene, pRSFduet-gpd1o-gpp2o-grol plasmid is constructed, and the map of pRSFduet-gpd1o-gpp2o-grol plasmid is shown in FIG. 1C.

The specific operation steps are as follows:

performing PCR amplification by taking an escherichia coli MG1655 genome as a template, designing an amplification Primer5 and a Primer6 by using bioinformatics software according to the escherichia coli molecular chaperone Gene sequence and the characteristics of multiple cloning sites on an expression vector pRSFdure-gpd 1o-gpp2 o-gril, and amplifying to obtain a molecular chaperone Gene sequence (Gene ID: 948665), wherein the nucleotide sequence of the molecular chaperone Gene sequence is shown as SEQ ID NO. 5; the primer sequences were as follows:

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 70 ℃ for 30sec; extension, 72 ℃ for 90sec (30 cycles); stopping extension at 72 ℃ for 10min; finally, the temperature is kept at 4 ℃.

The PCR product is subjected to 1% agarose gel electrophoresis detection analysis to obtain an electrophoresis band with the size of about 1.6kb, a target fragment is recovered through a gel recovery kit, a vector plasmid pRSFduet-gpd1o-gpp2o is subjected to double digestion by using Not I and Afl II, then the pRSFduet-gpd1o-gpp2o-grol plasmid is obtained through connection by a one-step cloning kit, the pRSFduet-gpd1o-gpp2o-grol plasmid is transferred into competent cells of escherichia coli BL21 (de 3) by a chemical transformation method, the competent cells are coated on a kanamycin solid LB plate containing 50 mu g/mL for overnight culture at 37 ℃, positive recombinants are picked up and stored at-80 ℃, and the recombinants are named as E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol; pRSFduet-gpd1o-gpp2o-grol plasmid was extracted from recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol using plasmid extraction kit.

Example 4: construction of plasmid pRSFduet-gpd1o-gpp2o-grol-gros

Cloning a chaperone 2 gene gros of Escherichia coli into pRSFduet-gpd1o-gpp2o-grol plasmid, wherein the chaperone 2 gene gros is located downstream of a promoter T7-promoter2 gpp2o gene, and constructing a map of pRSFduet-gpd1o-gpp2o-grol-gros plasmid as shown in FIG. 1D.

The specific operation steps are as follows:

performing PCR amplification by taking an escherichia coli MG1655 genome as a template, designing an amplification Primer7 and a Primer8 by using bioinformatics software according to the characteristics of a escherichia coli molecular chaperone 2 Gene gros sequence and a polyclonal site on a vector plasmid pRSFdure-gpd 1o-gpp2o-grol, and amplifying to obtain a molecular chaperone 2 Gene gros sequence (Gene ID: 948655), wherein the nucleotide sequence of the molecular chaperone 2 Gene gros sequence is shown as SEQ ID NO. 6; the primer sequences were as follows:

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 61 ℃ for 30sec; extending at 72 ℃ for 1min (30 cycles); stopping extension at 72 ℃ for 10min; finally, the temperature is kept at 4 ℃.

The PCR product is detected and analyzed by agarose gel electrophoresis with the concentration of 1%, an electrophoresis band with the size of about 300bp is obtained, a target fragment is recovered by a gel recovery kit, and after vector plasmid pRSFduet-gpd1o-gpp2o-grol is digested by Pac I and Avr II, pRSFduet-gpd1o-gpp2o-grol-gros plasmid is obtained by connecting the vector plasmid pRSFduet-gpd1o-gpp2o-grol-gros plasmid by a one-step cloning kit. Transferring pRSFduet-gpd1o-gpp2o-grol-gros plasmid into competent cells of escherichia coli BL21 (de 3) by a chemical transformation method, coating the competent cells on a solid LB plate containing 50 mug/mL kanamycin, culturing overnight at 37 ℃, picking up positive recombinants and preserving at-80 ℃, wherein the recombinants are named as E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros; pRSFdure-gpd 1o-gpp2o-grol-gros plasmid was extracted from recombinant E.coli BL21 (de 3)/pRSFdure-gpd 1o-gpp2o-grol-gros using plasmid extraction kit.

Example 5: construction of recombinant E.coli genetically engineered bacteria

(1) Extraction of recombinant plasmid

The recombinant plasmid pRSFduet-gpd1o-gpp2o was obtained from recombinant bacterium BL21 (de 3)/pRSFduet-gpd 1o-gpp2o stored in glycerol using a plasmid extraction kit.

(2) Transformation of competent cells to produce recombinant bacteria

Taking 10 mu L of recombinant plasmid pRSFduet-gpd1o-gpp2o extracted in the step (1), respectively taking 90 mu L of escherichia coli BL21 (star) and Rosetta competent cells, respectively mixing the recombinant plasmid pRSFduet-gpd1o-gpp2o with the competent cells, placing on ice for 30min, accurately heat-shocking for 45s at 42 ℃, placing on ice for 2-3min, taking out the competent cells, immediately adding the competent cells into 1mL of LB liquid medium, shaking and culturing for 1-2h at 37 ℃ for 200r/min, then respectively coating on LB solid medium plates containing 50 mu g/mL of kana resistance, 50 mu g/mL kana and 25ug/mL chloramphenicol resistance, and culturing overnight at 37 ℃ to obtain recombinant escherichia coli E.SFduet BL21 (star)/pRSFduet-gpd 1o-gpp2o and E.coli Rosetta/gpd 1o-gpp2o.

Single colonies were picked for colony PCR identification, E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o in example 2, E.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o in this example, and E.coli Rosetta/pRSFduet-gpd1o-gpp2o were subjected to PCR amplification using the corresponding primers (Primer 1 to 4) under the same PCR conditions as in examples 1 to 2, and the amplification products were subjected to PCR gel electrophoresis, the results of which are shown in FIGS. 2 and 3.

E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros single colonies of example 4 were amplified by PCR using the corresponding primers (primers 5 to 8) under the same conditions as in examples 3 to 4, and the amplified products were subjected to PCR gel electrophoresis, and the results are shown in FIG. 4.

As can be seen from FIGS. 2,3 and 4, the target bands appear in the gel electrophoresis patterns and are single, E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; the target fragment lengths of the E.coli Rosetta/pRSFduet-gpd1o-gpp2o single colonies were 1.1kb and 700bp, respectively.

The target fragment length of the E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros single colony is about 1.6kb and 200bp, and the colony is shown to be a positive clone, so that the positive recombinant escherichia coli E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o is obtained; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; e.coli Rosetta/pRSFduet-gpd1o-gpp2o; coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros and recombinant bacteria were stored at-80 ℃.

Example 6: construction of control strains

According to the methods described in examples 1 to 5, the genes encoding gpd1 and 3-phosphoglycerate of the non-optimized codon 3-phosphoglycerate dehydrogenase were amplified as gpp2, plasmids pRSFduet-gpd1 and pRSFduet-gpd1-gpp2 were constructed, and the plasmids pRSFduet-gpd1-gpp2 were transformed into E.coli BL21 (de 3) to obtain recombinant E.coli BL21 (de 3) pRSFduet-gpd1-gpp2 as a control strain.

Wherein, the amplification primer of the gene gpd1 is as follows:

primer9:5'-tcatcaccacagccaggatccATGTCTGCTGCTGCTGATAGATTAA-3' (containing BamHI cleavage site),

primer10:5'-gcattatgcggccgcaagcttCTAATCTTCATGTAGATCTAATTCTTCAATCA-3' (HindIII cleavage site);

the amplification primers for gene gpp2 were as follows:

primer11:5'-agatatacatatggcagatctATGGGATTGACTACTAAACCTCTATCTTT-3' (containing Bgl II cleavage site),

primer12:5'-ggtttctttaccagactcgagTTACCATTTCAACAGATCGTCCTT-3' (containing Xho I cleavage site).

Example 7: gene expression in recombinant E.coli

The positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o in example 5 was used; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; e.coli Rosetta/pRSFduet-gpd1o-gpp2o, E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros in example 4, inoculated in 50mL of liquid LB medium containing 50. Mu.g/mL kanamycin at an inoculum size of 2%, and activated at 37℃for 12h at 200 rpm; 1mL of the activated strain is inoculated into 100mL of liquid LB medium containing 50 mug/mL kanamycin, and the culture is carried out at 37 ℃ under shaking at 200rpm until the bacterial liquid OD ₆₀₀ At 0.8, 0.5mM IPTG was added and induction was performed at 20℃for 12 hours.

After fermentation, taking 100mL of fermentation liquor, centrifuging at 12000rpm and 4 ℃ for 10min, collecting thalli, and adding 10mL of phosphate buffer solution (pH 7.4) to resuspend the thalli; ultrasonic disruption of the somatic cells: ultrasonic treatment for 4s at intervals of 6s and 300W for 30min to obtain crude enzyme liquid; the obtained crude enzyme solution was subjected to SDS-PAGE electrophoresis to determine the size of the target protein, and the results are shown in FIG. 5.

Wherein the recombinant bacterium E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; the 3-phosphoglyceride dehydrogenase protein expressed by E.coli Rosetta/pRSFduet-gpd1o-gpp2o is about 27.8kDa and the 3-phosphoglyceride dehydrogenase protein is about 42.8kDa.

Example 8: glycerol detection

The positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o in example 5 was used; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; e.coli Rosetta/pRSFduet-gpd1o-gpp2o, E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol in example 3, E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros in example 4, and control strain E.coli BL21 (de 3) pRSFduet-gpd1-gpp2 in example 6 were activated on LB solid medium, and the activated single colonies were picked up and inoculated into seed medium, and cultured at 37℃and 200rpm for 12h to obtain seed liquid; the seed solution was inoculated into a conical flask containing 100mL of fermentation medium at an inoculum size of 2%, and fermented to 70 hours, and the residual glucose amount and the glycerol yield were measured by high performance liquid chromatography, and the fermentation results are shown in FIGS. 6, 7 and 8.

The positive recombinant E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o in example 5 was used; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; e, activating the coli Rosetta/pRSFduet-gpd1o-gpp2o on LB solid culture medium respectively, picking the activated single colony, inoculating the single colony into a seed culture medium, and culturing for 12 hours at 37 ℃ and 200rpm to obtain seed liquid; the seed liquid is inoculated into a 1L automatic stirring fermentation tank according to the inoculation amount of 10 percent, air is introduced into the fermentation tank at the rotating speed of 200r/min so as to maintain the aerobic fermentation environment, 2.5mol/L potassium hydroxide is automatically added to adjust the pH value of the fermentation liquid to 7.0, the fermentation temperature is maintained to be 37 ℃, and the fermentation time is 70h.

Wherein, the fermentation medium composition (/ L): mgSO (MgSO) ₄ ·7H ₂ O 0.48g，NaH ₂ PO4 6.0g，CaCl ₂ 0.0111g，NH ₄ Cl2.0g，NaCl 0.5g，KH ₂ PO ₄ 3.0g, yeast extract powder 5.0g and glucose 40g/L. And respectively taking the fermentation liquid after the fermentation is finished, and measuring the content of glycerol and glucose in the fermentation liquid by adopting a liquid chromatography method.

The liquid chromatography conditions were: SHIMADZU island body fluid phase chromatograph, RID differential detector, agilent 1260 definition II. Chromatographic conditions: column temperature 26 ℃,75% acetonitrile as mobile phase, and each sample injection amount is 10 mu L. The retention time of glycerol was 5.5min and the retention time of glucose was 10.9min, and the concentration of glycerol and glucose was calculated by the external standard method, and the results are shown in FIGS. 9, 10 and 11.

As shown in FIG. 6, the glycerol yield of the genetically engineered bacteria expressing the optimized codons of the invention, in which the coding genes of the 3-phosphoglycerate dehydrogenase are gpd1o and the coding genes of the 3-phosphoglycerate enzyme are gpp2o, is about 0.455g/L, which is significantly higher than that of the genetically engineered bacteria expressing the original genes of the 3-phosphoglycerate dehydrogenase and the 3-phosphoglycerate enzyme, and the glycerol yield of E.coli BL21 (de 3) pRSFduet-gpd1-gpp2 is about 0.118g/L, which is 3 times higher than that of the control strain.

As can be seen from FIG. 7, the effect of adding molecular chaperones was compared, and the genetically engineered bacterium E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o was subjected to shaking fermentation for 70 h; e.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol; the highest glycerol production concentrations of E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o-grol-gros were 0.175g/L,1.708/L and 4.425/L, respectively, and were 25 times that of the non-inserted chaperone strain E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o.

As can be seen from FIG. 8, comparing the effect of replacing the host bacteria, the genetically engineered bacteria E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o after shaking fermentation for 70 h; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; the highest glycerol production concentration of the E.coli Rosetta/pRSFduet-gpd1o-gpp2o can reach 0.163g/L,5.318g/L and 6.816g/L respectively, and the conversion rate of glucose to glycerol reaches 0.326%,10.636% and 13.632%, respectively, and compared with the shake flask fermentation of the control strain E.coli BL21 (de 3)/pRSFduet-gpd 1-gpp2 expressing non-optimized codons, the glycerol yield is improved by 58 times.

As can be seen from FIGS. 9, 10 and 11, the genetically engineered bacterium E.coli BL21 (de 3)/pRSFduet-gpd 1o-gpp2o after aerobic fermentation in a 1L mini-fermenter for 70 h; e.coli BL21 (star)/pRSFduet-gpd 1o-gpp2o; the highest yields of E.coli Rosetta/pRSFduet-gpd1o-gpp2o glycerol were 4.741g/L,8.958g/L and 10.897g/L, respectively, with glucose to glycerol conversions of 14.78%,27.39% and 33.4%, respectively.

Meanwhile, the results show that the invention can be implemented by optimizing codons of two glycerol-producing key enzyme genes from saccharomyces cerevisiae and selecting proper host bacteria for fermentation, and the results are obvious.

SEQUENCE LISTING

<110> Qilu university of industry

<120> genetically engineered bacterium for efficiently producing glycerol, construction method and application thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1176

<212> DNA

<213> Saccharomyces cerevisiae S288C

<400> 1

atgtctgctg ctgctgatag attaaactta acttccggcc acttgaatgc tggtagaaag 60

agaagttcct cttctgtttc tttgaaggct gccgaaaagc ctttcaaggt tactgtgatt 120

ggatctggta actggggtac tactattgcc aaggtggttg ccgaaaattg taagggatac 180

ccagaagttt tcgctccaat agtacaaatg tgggtgttcg aagaagagat caatggtgaa 240

aaattgactg aaatcataaa tactagacat caaaacgtga aatacttgcc tggcatcact 300

ctacccgaca atttggttgc taatccagac ttgattgatt cagtcaagga tgtcgacatc 360

atcgttttca acattccaca tcaatttttg ccccgtatct gtagccaatt gaaaggtcat 420

gttgattcac acgtcagagc tatctcctgt ctaaagggtt ttgaagttgg tgctaaaggt 480

gtccaattgc tatcctctta catcactgag gaactaggta ttcaatgtgg tgctctatct 540

ggtgctaaca ttgccaccga agtcgctcaa gaacactggt ctgaaacaac agttgcttac 600

cacattccaa aggatttcag aggcgagggc aaggacgtcg accataaggt tctaaaggcc 660

ttgttccaca gaccttactt ccacgttagt gtcatcgaag atgttgctgg tatctccatc 720

tgtggtgctt tgaagaacgt tgttgcctta ggttgtggtt tcgtcgaagg tctaggctgg 780

ggtaacaacg cttctgctgc catccaaaga gtcggtttgg gtgagatcat cagattcggt 840

caaatgtttt tcccagaatc tagagaagaa acatactacc aagagtctgc tggtgttgct 900

gatttgatca ccacctgcgc tggtggtaga aacgtcaagg ttgctaggct aatggctact 960

tctggtaagg acgcctggga atgtgaaaag gagttgttga atggccaatc cgctcaaggt 1020

ttaattacct gcaaagaagt tcacgaatgg ttggaaacat gtggctctgt cgaagacttc 1080

ccattatttg aagccgtata ccaaatcgtt tacaacaact acccaatgaa gaacctgccg 1140

gacatgattg aagaattaga tctacatgaa gattag 1176

<210> 2

<211> 1176

<212> DNA

<213> Saccharomyces cerevisiae S288C

<400> 2

atgagcgcgg cggctgaccg tctgaacctg acctctggtc acctgaacgc gggtcgtaaa 60

cgttcttctt ctagcgtttc tctgaaagcg gcagaaaaac cgttcaaagt taccgtgatc 120

ggttccggta actggggcac caccatcgcg aaagttgttg cggaaaactg caaaggttac 180

ccggaagtgt ttgctccgat cgttcagatg tgggttttcg aagaagaaat caacggtgaa 240

aaactgaccg aaatcatcaa cacccgtcac cagaacgtta aatacctgcc gggtatcacc 300

ctgccggaca acctggtggc gaacccggat ctgatcgata gcgttaaaga tgttgatatc 360

atcgttttca acatcccgca ccagttcctg ccgcgtatct gctctcagct gaaaggtcac 420

gttgattctc acgttcgcgc gattagctgc ctgaaaggct ttgaagtggg tgcgaaaggc 480

gttcagctgc tgtctagcta cattaccgaa gaactgggca tccagtgcgg cgcgctgtct 540

ggcgcaaaca tcgcgaccga agttgcgcag gaacactggt ctgaaactac cgtggcttac 600

cacatcccga aagatttccg tggtgaaggt aaagatgttg atcacaaagt tctgaaagcg 660

ctgttccacc gtccgtactt ccatgtttcc gttatcgaag atgttgctgg catctctatc 720

tgcggcgcgc tgaaaaacgt tgttgcgctg ggctgtggtt tcgttgaagg cctgggttgg 780

ggcaacaacg cttctgctgc gatccagcgt gttggcctgg gtgaaatcat ccgtttcggc 840

cagatgttct tcccggaaag ccgtgaagaa acctactacc aggaatccgc tggcgttgct 900

gacctgatca ccacctgcgc gggtggccgt aacgttaaag ttgcgcgtct gatggcgacc 960

tctggtaaag atgcgtggga atgcgaaaaa gaactgctga acggtcagtc tgcacagggc 1020

ctgattacct gcaaagaagt tcacgaatgg ctggaaacct gcggctccgt tgaagatttc 1080

ccgctgttcg aagcggtgta ccagatcgtt tataacaact acccgatgaa aaacctgccg 1140

gatatgatcg aagaactgga tctgcacgag gactaa 1176

<210> 3

<211> 753

<212> DNA

<213> Saccharomyces cerevisiae S288C

<400> 3

atgggattga ctactaaacc tctatctttg aaagttaacg ccgctttgtt cgacgtcgac 60

ggtaccatta tcatctctca accagccatt gctgcattct ggagggattt cggtaaggac 120

aaaccttatt tcgatgctga acacgttatc caagtctcgc atggttggag aacgtttgat 180

gccattgcta agttcgctcc agactttgcc aatgaagagt atgttaacaa attagaagct 240

gaaattccgg tcaagtacgg tgaaaaatcc attgaagtcc caggtgcagt taagctgtgc 300

aacgctttga acgctctacc aaaagagaaa tgggctgtgg caacttccgg tacccgtgat 360

atggcacaaa aatggttcga gcatctggga atcaggagac caaagtactt cattaccgct 420

aatgatgtca aacagggtaa gcctcatcca gaaccatatc tgaagggcag gaatggctta 480

ggatatccga tcaatgagca agacccttcc aaatctaagg tagtagtatt tgaagacgct 540

ccagcaggta ttgccgccgg aaaagccgcc ggttgtaaga tcattggtat tgccactact 600

ttcgacttgg acttcctaaa ggaaaaaggc tgtgacatca ttgtcaaaaa ccacgaatcc 660

atcagagttg gcggctacaa tgccgaaaca gacgaagttg aattcatttt tgacgactac 720

ttatatgcta aggacgatct gttgaaatgg taa 753

<210> 4

<211> 753

<212> DNA

<213> Saccharomyces cerevisiae S288C

<400> 4

atgggtctga ccaccaaacc gctgagcctg aaagttaacg cggcgctgtt cgatgttgat 60

ggcaccatca tcatcagcca gccggcgatc gcggcgttct ggcgtgattt cggtaaagat 120

aaaccgtact tcgatgcgga acacgttatc caggtttctc acggttggcg taccttcgat 180

gcgatcgcga aattcgctcc ggatttcgcg aacgaagaat acgttaacaa actggaagcg 240

gaaatcccgg ttaaatacgg tgaaaaatct atcgaagttc cgggtgcggt taaactgtgc 300

aacgcgctga acgcgctgcc gaaagaaaaa tgggcggttg cgacctctgg tacccgtgat 360

atggcgcaga aatggttcga acacctgggc atccgtcgtc cgaaatactt catcaccgcg 420

aacgatgtta aacagggcaa accgcacccg gaaccgtacc tgaaaggccg taacggcctg 480

ggttacccga tcaacgaaca ggatccgagc aaaagcaaag ttgttgtttt cgaagatgcg 540

ccggcgggca tcgcggcggg caaagcggcg ggctgcaaaa tcatcggcat cgcgaccacc 600

ttcgatctgg atttcctgaa agaaaaaggc tgcgatatca tcgttaaaaa ccacgaatct 660

atccgtgttg gtggctacaa cgcggaaacc gatgaagttg aattcatctt cgatgattac 720

ctgtacgcga aagatgatct gctgaaatgg taa 753

<210> 5

<211> 1647

<212> DNA

<213> Escherichia coli MG1655

<400> 5

atggcagcta aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta 60

aacgtactgg cagatgcagt gaaagttacc ctcggtccaa aaggccgtaa cgtagttctg 120

gataaatctt tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc 180

gaactggaag acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctctaaa 240

gcaaacgacg ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc 300

actgaaggtc tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc 360

gacaaagcgg ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc atgctctgac 420

tctaaagcga ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa 480

ctgatcgctg aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt 540

accggtctgc aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg 600

tctccttact tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc 660

ctgctggctg acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt 720

gccaaagcag gcaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca 780

actctggttg ttaacaccat gcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc 840

ttcggcgatc gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg 900

atctctgaag agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct 960

aaacgtgttg tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct 1020

gcaatccagg gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac 1080

gaccgtgaaa aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa 1140

gtgggtgctg ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg 1200

cacgcgaccc gtgctgcggt agaagaaggc gtggttgctg gtggtggtgt tgcgctgatc 1260

cgcgtagcgt ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc 1320

aaagttgcac tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa 1380

gaaccgtctg ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca 1440

gcaaccgaag aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact 1500

cgttctgctc tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgaatgcatg 1560

gttaccgacc tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc 1620

atgggtggca tgggcggcat gatgtaa 1647

<210> 6

<211> 294

<212> DNA

<213> Escherichia coli MG1655

<400> 6

atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60

tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120

ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180

gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240

gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gtaa 294

Claims

1. A genetic engineering bacterium for efficiently producing glycerol is characterized in that the genetic engineering bacterium simultaneously enhances and expresses 3-phosphoglycerate dehydrogenase and 3-phosphoglycerate enzyme after optimizing codons, and is characterized in that the 3-phosphoglycerate dehydrogenase is 3-phosphoglycerate dehydrogenase from saccharomyces cerevisiae, and the coding gene isgpd1oThe nucleotide sequence is shown as SEQ ID NO. 2; the 3-phosphoglycerase is 3-phosphoglycerase from Saccharomyces cerevisiae, and the coding gene isgpp2oThe nucleotide sequence is shown as SEQ ID NO. 4;

the host strain isE.coliBL21(de3)、E.coliBL21(star) Or (b)E.coliRosetta; the expression vector used for constructing the genetically engineered bacterium has the structure that the 3-phosphoglycerate dehydrogenase gene is positioned at the upstream of the 3-phosphoglycerate enzyme;

the genetically engineered bacterium is recombinantE.coliBL21(de3) During the process, inserting molecular chaperone 1 and molecular chaperone 2 into the genetically engineered bacteria; said chaperone 1 is derived from E.coli and its coding gene isgrolThe nucleotide sequence is shown as SEQ ID NO. 5; said chaperone 2 is derived from E.coli and its coding gene isgrosThe nucleotide sequence is shown as SEQ ID NO. 6; the molecular chaperone 1 in the expression vector used for constructing the genetically engineered bacteria is positioned at the downstream of the 3-phosphoglycerate dehydrogenase,upstream of the 3-phosphoglycerase; chaperone 2 is located downstream of 3-phosphoglycerase.

2. The method for constructing genetically engineered bacteria of claim 1, comprising the steps of:

(1) Saccharomyces cerevisiae 3-phosphoglycerate dehydrogenase genegpd1oInserting into vector plasmid pRSFduet to construct expression vector pRSFduet-gpd1o；

(2) Saccharomyces cerevisiae 3-phosphoglycerase genegpp2oInsertion of vector plasmid pRSFduet-gpd1oIn the process, an expression vector pRSFduet-gpd1o-gpp2o；

(3) Chaperone 1 gene of colibacillusgrolInsertion of vector plasmid pRSFduet-gpd1o-gpp2oIn the process, an expression vector pRSFduet-gpd1o-gpp2o-grol；

(4) Chaperone 2 gene g of Escherichia colirosInsertion of vector plasmid pRSFduet-gpd1o-gpp2o-grolIn the process, an expression vector pRSFduet-gpd1o-gpp2o-grol-gros；

(5) Expression vector pRSFduet-gpd1o-gpp2oRespectively transformed into Escherichia coliE.coli BL21(star) AndE.coliin Rosetta, the expression vector pRSFduetgpd1o-gpp2o-grol-grosTransformation into E.coliE.coliBL21(de3) Selecting positive recombinants to obtain genetically engineered bacteriaE.coliBL21 (de3)/pRSFduet-gpd1o-gpp2o- grol-gros、E.coliBL21(star)/pRSFduet-gpd1o-gpp2oAndE.coliRosetta/pRSFduet-gpd1o-gpp2o。

3. the method for constructing genetically engineered bacteria of claim 2, comprising the steps of:

(1) PCR amplification is carried out by taking S288C genome of saccharomyces cerevisiae as a template to obtain 3-phosphoglycerate dehydrogenasegpd1oThe sequences, PCR primer sequences were as follows:

Primer1:5′- cgcctgcaggtcgacaagcttATGAGCGCGGCGGCTGAC -3', containingBamHI enzyme cutting site;

primer2:5'-agctgccatctccttgcggccgcTTAGTCCTCGTGCAGATCCAGTT-3', containingHind III, enzyme cutting site;

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 60 ℃ for 40sec; extending at 72 ℃ for 30sec,30 cycles; stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet was usedHindIIINotAfter I double digestion, pRSFduet is obtained by one-step cloning kit connectiongpd1oA plasmid;

(2) PCR amplification is carried out by taking S288C genome of saccharomyces cerevisiae as a template to obtain 3-phosphoglyceride enzyme genegpp2oThe sequences, PCR primer sequences were as follows:

primer3:5'-taagaaggagatatacatatg ATGGGTCTGACCACCAAACCG-3', containingNdeI enzyme cutting site;

primer4:5'-gccgatatccaattgagatct TTACCATTTCAGCAGATCATCTTTCG-3', containingBglII enzyme cutting sites;

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 60 ℃ for 30sec; extension, 72 ℃ 90sec,30 cycles; stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet obtained in step (1) is used for preparing the plasmid pRSFduetgpd1oRespectively usingNdeI andBglII, after double enzyme digestion, pRSFduet is obtained by one-step cloning kit connectiongpd1o-gpp2oA plasmid;

(3) PCR amplification is carried out by taking the escherichia coli MG1655 genome as a template to obtain a molecular chaperone 1 genegrolThe sequences, PCR primer sequences were as follows:

primer5:5'-caggtcgacaagcttgcggccgc AAGGAGATGGCAGCTAAAGACGTAAAATTCG-3', containingNotI enzyme cutting site;

primer6:5'-ttactttctgttcgacttaag TTACATCATGCCGCCCATG-3', containingAflII enzyme cutting sites;

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 65 ℃ for 30sec; extending at 72 ℃ for 45s,30 cycles; stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet obtained in step (2) is used for preparing the plasmid pRSFduetgpd1o-gpp2oBy usingNotI andAflII, after double enzyme digestion, pRSFduet is obtained by one-step cloning kit connectiongpd1o-gpp2o-grolA plasmid;

(4) PCR amplification is carried out by taking the escherichia coli MG1655 genome as a template to obtain a molecular chaperone 2 genegrosThe sequences, PCR primer sequences were as follows:

primer7:5'-gtctactagcgcagcttaattaaAAGGAGATGAATATTCGTCCATTGCATGAT-3', containingPacI enzyme cutting site;

primer8:5'-cagcggtggcagcagcctaggTTACGCTTCAACAATTGCCAGA-3', containingAvrII enzyme cutting sites;

PCR amplification procedure: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃; annealing at 65 ℃ for 30sec; extension, 15s at 72℃for 30 cycles; stopping extension at 72 ℃ for 10min; finally preserving heat at 4 ℃;

the vector plasmid pRSFduet obtained in step (3) is used for preparing the plasmid pRSFduetgpd1o-gpp2o-grolBy usingPacI andAvrII, after double enzyme digestion, pRSFduet is obtained by one-step cloning kit connectiongpd1o-gpp2o-grol-grosA plasmid;

(5) pRSFduet obtained in step (2)gpd1o-gpp2oPlasmid is respectively and chemically transformed into competent cells BL21 #, which is a competent cell of the escherichia colistar) And Rosetta, pRSFduet obtained in step (4)gpd1o-gpp2o-grol-grosPlasmid chemical transformation of competent cell BL21de3) Selecting positive recombinants to obtain genetically engineered bacteriaE.coliBL21 (de3)/pRSFduet-gpd1o-gpp2o-grol-gros、E.coliBL21(star)/pRSFduet-gpd1o-gpp2oAndE.coliRosetta/pRSFduet-gpd1o-gpp2o。

4. the use of the genetically engineered bacterium of claim 1 in the production of glycerol.

5. The use according to claim 4, wherein the glycerol is produced by fermentation using glucose as a substrate by the following process:

activating the genetically engineered bacterium in an LB solid medium, picking an activated single colony, inoculating the single colony into an LB liquid medium, culturing for 12 hours at 37 ℃ and 200r/min, inoculating the single colony into a fermentation medium according to an inoculum size of 2% -10%, and fermenting at 37 ℃ to produce glycerol;

wherein, the fermentation medium comprises: mgSO (MgSO) ₄ ·7H ₂ O 0.48g/L，NaH ₂ PO ₄ 6.0g/L，CaCl ₂ 0.0111g/L，NH ₄ Cl 2.0g/L， NaCl 0.5g/L，KH ₂ PO ₄ 3.0g/L, 5.0g/L of yeast extract powder and 40g/L of glucose.