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A method for improving the efficiency of Escherichia coli to synthesize fumaric acid

A technology of Escherichia coli and fumaric acid, applied in the field of synthetic biology to achieve the effect of improving efficiency

Active Publication Date: 2020-02-11
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to solve the efficiency problem of fumaric acid synthesis in escherichia coli

Method used

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  • A method for improving the efficiency of Escherichia coli to synthesize fumaric acid
  • A method for improving the efficiency of Escherichia coli to synthesize fumaric acid
  • A method for improving the efficiency of Escherichia coli to synthesize fumaric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example illustrates the construction process of the recombinant vector pTRC99A-mdh-linker-st-sc-linker-ppc-linker-sc-st-linker-fumR of the enzymatic system for the synthesis of fumaric acid containing a specific tag. The Rhizopus oryzae strain used has a preservation number of CCTCC: M207066

[0036] (1) Extract Rhizopus oryzae mRNA, reverse transcribe to obtain cDNA, design primer 1 and primer 2, primer 3 and primer 4, primer 5 and primer 6 respectively, and obtain the product pyruvate dehydrogenase gene pyc respectively by PCR method, Malate dehydrogenase gene mdh and fumarase gene fumR, gel recovery PCR products;

[0037] (2) Using the label sc gene as a template, design primers P1 and P2, and obtain the sc PCR product with a linker at the C-terminal by PCR, and name it sc-linker;

[0038] (3) The obtained pyc product was used as a template, and primers P3 and P4 were designed to obtain the PCR product of the pyruvate dehydrogenase gene pyc with a linker at the...

Embodiment 2

[0081] This example illustrates the effect of a target vector containing a single gene on the production of fumaric acid

[0082] Pick a single colony of the target carrier strain containing only a single gene pyc in the ultra-clean bench and put it in 50 mL of fresh liquid LB medium as the seed liquid, and add 50 uL of AMP with a final concentration of 50 mg / mL, culture at 37 °C, 200 rpm 10-12 h, according to the inoculation amount of 1% (volume ratio), receive 50 mL of fresh fermentation medium (plus 50 uLAMP), incubate at 37 ℃, 200 rpm for 12 h, then centrifuge at 8000 rpm for 10 min, take the supernatant, and finally Utilize HPLC to carry out content determination to fumaric acid, its output reaches 1.372 g / L ( image 3 ).

Embodiment 3

[0084] This implementation case illustrates the impact of the target carrier strain on the production of fumaric acid

[0085] Pick a single colony of the original strain (as a control) target tag carrier strain in the ultra-clean bench and put it in 50 mL of fresh liquid LB medium as the seed liquid, and add 50 uL of AMP with a final concentration of 50 mg / mL, 37 ℃, 200 rpm, cultured for 10-12 h, received 50 mL of fresh fermentation medium (plus 50 uLAMP) according to the inoculation amount of 1% (volume ratio), and cultured at 200 rpm at 37 °C for 16 h, every 3 hours during the middle process Take a sample once, then centrifuge at 8000 rpm for 10 min, take the supernatant, and the final fumaric acid yield reaches 6.772 g / L ( Figure 4 ).

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Abstract

The invention provides a method for improving efficiency of escherichia coli on synthesizing fumaric acid. Specificity labels are added on two ends of three key enzymes (pyruvate dehydrogenase, malic dehydrogenase and fumarase) synthesized by the fumaric acid, so that covalent binding of the three enzymes is realized, a substrate channel is shortened, and the synthetic efficiency of the fumaric acid in the escherichia coli is promoted.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a method of synthetic biology, in particular to adopting genetic engineering technical means to reduce substrate channels and improve the catalytic efficiency of key enzymes in fumaric acid synthesis pathways in Escherichia coli. Background technique [0002] As an intermediate metabolite of the tricarboxylic acid cycle, fumaric acid is an important bulk chemical product. It is widely used in coatings, resins, medicines, plasticizers and other fields. It is listed as 12 priority biological species by the US Department of Energy. One of the base platform compounds, the global demand reaches one million tons. At present, the chemical method is mainly used to synthesize fumaric acid in industry, but its production conditions are harsh, the catalyst is highly toxic, and the environment is seriously polluted; relatively speaking, the microbial fermentation method to synthesize fumaric acid has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/62C12N15/10C12N1/21C12P7/46
CPCC07K2319/00C12N9/0006C12N9/0008C12N9/88C12N15/10C12N15/62C12N15/70C12P7/46C12Y101/01037C12Y402/01002
Inventor 黄和彭程徐晴江凌李霜
Owner NANJING TECH UNIV
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