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Formic acid and CO2 autotrophic recombinant escherichia coli and construction method thereof

A technology for recombinant Escherichia coli and construction methods, applied in the directions of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of poor general performance, long operation cycle, high input cost, and achieve universality The effect of high, short cycle time and simple construction process

Pending Publication Date: 2021-03-12
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above method has obtained some formic acid and CO 2 Self-supporting recombinant Escherichia coli, but there are still problems of long operation period, high input cost and poor general performance

Method used

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  • Formic acid and CO2 autotrophic recombinant escherichia coli and construction method thereof
  • Formic acid and CO2 autotrophic recombinant escherichia coli and construction method thereof
  • Formic acid and CO2 autotrophic recombinant escherichia coli and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Modular plasmid construction of formic acid assimilation function

[0040] According to the gene sequence and map of the commercial plasmid pETDuet-1, the upstream primer pEM6-F carrying the SalI restriction site and the downstream primer pEM6-R carrying the AvrII restriction site were respectively designed, and the pETDuet-1 plasmid was obtained by PCR amplification The linearized fragment; the plasmid linearized fragment obtained by SalI / AvrII double digestion and the tac gene synthesis fragment (sequence is the sequence between AvrII and SalI restriction sites shown in SEQ ID NO.4), passed through T4 DNA ligase was used to ligate the digested plasmid linearized fragment and tac gene fragment at 16°C for 1 hour to transform Escherichia coli JM109 competent; finally, the plasmid containing pEM6tac ( SEQ ID NO.4 is the clone of the plasmid sequence), amplified and extracted for later use. According to Clostridium ljungdahlii ( Clostridium ljungdahlii ) of ...

Embodiment 2

[0043] Example 2: CO 2 Modular plasmid construction of assimilative function

[0044] The glycine lyase complex is generally considered to have a reverse catalytic function under the condition of sufficient reducing cofactor NAD(P)H, that is, to convert CO 2 , NH 4 + and formic acid assimilation product CH 2 - The ability of THF (methylenetetrahydrofolate) to convert to glycine. Therefore, the glycine lyase complex for CO 2 Assimilation plays a key role and is the rate-limiting step in achieving autotrophy in E. coli-dependent C1-pyruvate synthesis pathway.

[0045] According to the glycine lyase of Escherichia coli BL21(DE3) itself EcgcvTHP Gene cluster sequence (as shown in SEQ ID NO.3), respectively design gcvT-F and gcvP-R primers carrying NcoI / BamHI restriction sites (see Table 1), perform PCR amplification on the above sequence to obtain about 4500 bp Gene fragments; use restriction endonucleases NcoI / BamHI to double-enzyme digest the obtained PCR product and pAC...

Embodiment 3

[0048] Embodiment 3: the construction of recombinant escherichia coli BL21 (DE3)

[0049] Escherichia coli BL21(DE3) was used as the chassis bacteria, as the formic acid assimilation plasmid and CO 2 The transformation object of the assimilation plasmid. The preparation method of Escherichia coli competent refers to the routine molecular biology operation steps: first, use LB medium to culture cells at 37°C to a concentration of 0.4 OD 600 ; Secondly, the cells were collected under centrifugation conditions at 4°C and 4000 rpm, and then washed twice with de-cooled sterile ionized water; finally, diluted Escherichia coli with cooled sterile deionized water according to 1000:1, obtained for Transform competent cells with modular plasmids.

[0050] Take 100 ng of pEM6tac-fchA-fhs-folD and pACYC-EcgcvTHP-CbFDH1 plasmids, respectively, and add them to an electric shock cup containing 100 µL of freshly prepared competent Escherichia coli BL21(DE3), mix gently, and incubate on ice ...

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Abstract

The invention discloses formic acid and CO2 autotrophic recombinant escherichia coli and a construction method thereof, and belongs to the field of microbial metabolic engineering and the field of synthetic biology. According to the invention, the most common escherichia coli is used as a starting strain, and formic acid assimilated modular plasmids and CO2 assimilated modular plasmids are introduced to construct the escherichia coli which rapidly grows by using formic acid and CO2 as a unique carbon source. The whole construction process is simple, the period is short, and the universality ishigh. According to the recombinant bacterium, the level of an intracellular reduced cofactor NAD (P) H can be improved by virtue of a formate dehydrogenase mutant, so that the recombinant bacterium has the capability of promoting pyruvic acid synthesis from beginning to end by eating formic acid, and finally formic acid and CO2 autotrophic escherichia coli is obtained; the escherichia coli is inoculated to a culture medium taking formic acid and CO2 as a unique carbon source for fermentation culture, 0.9 OD600 biomass can be obtained at most within 56 hours, and the original strain hardly grows. The escherichia coli is methanoic acid and CO2 autotrophic escherichia coli with the highest growth speed under the condition of no assistance of gene knockout, long-term domestication and other means at present.

Description

technical field [0001] The invention discloses a kind of formic acid and CO 2 The self-supporting recombinant Escherichia coli and its construction method belong to the field of microbial metabolic engineering and synthetic biology. Background technique [0002] With the rapid development of social economy and the excessive development and utilization of fossil energy, a large amount of greenhouse gas emissions have brought a huge threat to environmental ecology, global climate, and human production and life. Carbon dioxide is the representative greenhouse gas with the largest emission, especially the carbon dioxide emitted by fossil fuels and industries is close to 40 billion tons per year. How to effectively manage and utilize carbon dioxide has become one of the most concerned scientific issues in the world today. [0003] CO 2 As one of the most basic carbon resources, it is an important precursor for the synthesis of other carbon-neutral compounds (such as glucose pr...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/52C12N15/53C12N15/60C12N15/70C12N15/66C12R1/19
CPCC12N9/00C12N9/0008C12N9/88C12N15/70C12N15/66C12Y102/01002
Inventor 方真沙冲张荣俞洋洋
Owner JIANGSU UNIV
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