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Genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as construction method and application thereof

A technology of high-density fermentation and genetically engineered bacteria, applied in the field of biochemical industry, can solve the problems of inability to reduce the accumulation of acetic acid, prolong the fermentation cycle, treat the symptoms but not the root cause, and achieve the effects of shortening the production cycle, reducing production costs, and strengthening separation and extraction

Active Publication Date: 2015-04-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the optimization of medium components can only slow down the negative effect of acetic acid on the expression of the target product, but cannot reduce the accumulation of acetic acid, and the change of components will often increase the cost, and will also have an adverse effect on the downstream purification process; reduce the specific growth rate Although the rate can reduce acetic acid, it will also delay the growth of cells and prolong the fermentation period; the dialysis culture method can only treat the symptoms but not the root cause, and cannot avoid the formation of acetic acid, and part of the medium will be lost in the process of removing acetic acid

Method used

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  • Genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as construction method and application thereof
  • Genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as construction method and application thereof
  • Genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of Genetically Engineered Bacteria Realizing High-Density Fermentation and Co-production of (R,R)-2,3-Butanediol

[0041] 1. Cloning of α-acetolactate synthase gene alsS, α-acetolactate decarboxylase gene budA and (R,R)-2,3-butanediol dehydrogenase gene bdhA

[0042] (1) Cloning of alsS gene encoding α-acetolactate synthase

[0043] Synthetic primers were designed according to the alsS gene sequence in Klebsiella pneumoniae (K.pneumoniae) published by Genebank:

[0044] P1: 5'-GATATGGACAAACAGTATCCGGT-3'

[0045] P2: 5'-GCGTTACAGAATCTGACTCAGAT-3'

[0046] Using the genomic DNA of K.pneumoniae CICC10011 as a template, using primers P1 and P2, Takara high-fidelity enzyme HS (Premix) PCR amplified alsS gene.

[0047] PCR reaction system: HS (Premix) 25 μL, primer P10.3 μL, primer P20.3 μL, K.pneumoniae CICC10011 genome 0.4 μL, ddH 2 O 24 μL.

[0048] PCR reaction conditions: 98°C for 10s, 62°C for 15s, 72°C for 1min45s, cycle 30 times; 72°C f...

Embodiment 2

[0169] Example 2 High-density fermentation implementation of the genetically engineered bacteria-recombinant Escherichia coli LG-R in Example 1 to produce (R,R)-2,3-butanediol strains: Escherichia coli MG1655, Escherichia coli LG-R;

[0170] Seed medium (g / L): peptone 10; yeast extract 5; NaCl 10.

[0171] Fermentation medium (g / L): glucose 80; K 2 HPO 4 ·3H 2 O 13.7; KH 2 PO 4 2.0; (NH 4 ) 2 HPO 4 3.3; (NH 4 ) 2 SO 4 6.6; MgSO 4 ·7H 2 O 0.25; FeSO 4 ·7H 2 O 0.05; ZnSO 4 ·7H 2 O 0.01; MnSO 4 ·H 2 O 0.01; CaCl 2 0.01; EDTA 0.05.

[0172] Fermentation conditions:

[0173] Preparation of seed solution: transfer the strains preserved in glycerol tubes to fresh LB liquid medium, activate at 37°C and 200rpm for 16h; connect 1 ring of activated strains to LB solid medium with corresponding antibiotics, and culture at 37°C for 12h Pick a single bacterium colony from the fresh flat plate in the 250mL Erlenmeyer flask that 50mL seed medium is housed, cultivate 12...

example 3

[0179] Example 3 realizes high-density fermentation and co-production of meso-2, the construction of genetically engineered bacteria of 3-butanediol

[0180] 1. Amplify the operon gene fragment alsSbudAbudC of the gene encoding α-acetolactate synthase, the gene encoding α-acetolactate decarboxylase and the gene encoding meso-2,3-butanediol dehydrogenase

[0181] Using the genomic DNA of K.pneumoniae CICC10011 as a template, using primers P15 and P16, Takara high-fidelity enzyme HS (Premix) was used for PCR amplification, and the amplified fragments included: promoter, RBS site, alsS, budA, budC and terminator.

[0182] P15: 5'-ACCTATTGACAATTAAAGGCTAAAATGCTATAATTCCACAAATCGGAGGATATACATATGAATCATTCTGCTGAATG-3'

[0183] P16: 5'-AAAAGGCCATCCGTCAGGATGGCCTTCTTTAGTTAAATACCATCCCGC-3'

[0184] PCR reaction system: HS (Premix) 25μL, primer P150.3μL, primer P160.3μL, K.pneumoniae CICC10011 genome 0.4μL, ddH 2 O 24 μL.

[0185] PCR reaction conditions: 98°C for 10s, 58°C for 15s, 72°...

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Abstract

The invention discloses genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as a construction method and an application thereof. The genetically engineered bacteria are constructed by integrating three key enzyme genes such as alpha-acetolactate synthetase encoding genes, alpha-acetolacetate decearboxylase encoding genes and 2,3-butanediol dehydrogenase encoding genes onto Escherichia coli chromosomes in a 2,3-butanediol synthesis path. According to the strain fermentation process, the content of the byproduct acetic acid is reduced, so that high-density fermentation can be realized, and 2,3-butanediol with high additional value is co-produced. In addition, the invention also discloses a method for realizing high-density fermentation co-production of other compounds and 2,3-butanediol genetically engineered bacteria and an application thereof. The 2,3-butanediol can be produced by virtue of high-density fermentation, and polyhydroxyalkanoates or functional proteins also can be co-produced, so that low-cost and high-efficiency co-production of the polyhydroxyalkanoates or functional proteins and the 2,3-butanediol is realized, and the genetically engineered bacteria have important industrial application values.

Description

technical field [0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a genetically engineered bacterium for realizing high-density fermentation of Escherichia coli and co-production of 2,3-butanediol and its construction method and application. Background technique [0002] Escherichia coli, as the first strain used in industrial production, has become a strain due to its simple structure, clear genetic background, easy culture, fast growth, high expression level of target genes, short culture period, and strong anti-pollution ability. The most commonly used prokaryotic expression system. With the deepening of research in the fields of metabolic engineering and fermentation engineering, Escherichia coli is used to ferment and produce a variety of high-value-added polymers, such as polyhydroxyalkanoate and various functional proteins, such as protease, insulin, and human-like collagen etc. are widely used. [0003] Hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/18C12N9/50C12P21/00C12R1/19
CPCC12N1/20C12N15/70C12P7/18C12P7/6409C12P21/02C12R2001/19C12N1/205
Inventor 纪晓俊刘陆罡黄和童颖佳沈梦秋聂志奎
Owner NANJING UNIV OF TECH
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