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Construction method of biosensor capable of dynamically promoting high yield of 3HP (3-hydroxypropionic acid)

A technology of biosensor and construction method, applied in the field of biosensor construction to dynamically promote the high production of 3-hydroxypropionic acid, capable of solving problems such as bacterial toxicity

Pending Publication Date: 2017-11-28
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The malonyl-CoA pathway uses acetyl-CoA synthesized from cheap carbon sources as the starting compound, and overexpression of the malonyl-CoA carboxylase gene acc can increase the production of 3HP, but excessive synthesis of malonyl-CoA has a negative effect on the bacterial produce poison

Method used

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  • Construction method of biosensor capable of dynamically promoting high yield of 3HP (3-hydroxypropionic acid)
  • Construction method of biosensor capable of dynamically promoting high yield of 3HP (3-hydroxypropionic acid)
  • Construction method of biosensor capable of dynamically promoting high yield of 3HP (3-hydroxypropionic acid)

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Experimental program
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Effect test

specific Embodiment approach 1

[0023] Embodiment 1: In this embodiment, a biosensor construction method for dynamically promoting high yield of 3HP is carried out according to the following steps:

[0024] 1. Construction of biosensor dynamic synthesis of 3HP engineering bacteria:

[0025] 1. Construction of the plasmid pE7a-acc that converts acetyl-CoA to malonyl-CoA, the plasmid pS2c-mcr that converts malonyl-CoA into 3HP, and the biosensor plasmid pBFR1k-lacI-8FapR designed based on the transcription factor FapR , transferred to E.coli BL21(DE3) competent cells by electroporation, and then screened and cultured on the resistant medium to obtain engineering bacteria E.coli3HP-0, and its preservation number is CGMCC No.14224.

[0026] 2. Activation of 3HP-producing engineering bacteria:

[0027] The constructed biosensor dynamically synthesized 3HP engineered bacteria in LB medium supplemented with appropriate antibiotics, at 37°C, 180rpm for the first overnight culture. The overnight LB culture was inoc...

specific Embodiment approach 2

[0032] Specific embodiment two: the difference between this embodiment and specific embodiment one is: use 0.1mM-0.4mM and 0.6mM-1mM IPTG induction culture in step three. Others are the same as the first embodiment.

specific Embodiment approach 3

[0033] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that in step 3, 0-0.001% and 0.1%-1% L-Arabinose is used to induce culture. Others are the same as those in the first or second embodiment.

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Abstract

The invention discloses a construction method of a biosensor capable of dynamically promoting high yield of 3HP (3-hydroxypropionic acid) and aims to eliminate the contradiction that in the 3HP synthesis process, an overexpressed malonyl CoA carboxylase gene (acc) can increase the yield of 3HP but excessive synthesized malonyl CoA poisons bacteria. A transcription factor FapR is sensitive to malonyl CoA, a negative feedback regulatory pathway is designed by use of the FapR and a promoter sequence to regulate transcription according to the concentration of malonyl CoA, the synthesis and transformation rate of malonyl CoA is controlled, and the yield of 3HP is increased while poisoning to bacterium cells is avoided. Experiments at the early stage discover that the malonyl CoA sensing controller can dynamically regulate the pathway and increase the yield of 3HP.

Description

technical field [0001] The present invention designs a biosensor construction method for dynamically promoting high production of 3-hydroxypropionic acid (3-Hydroxypropionic acid, 3HP). Using biosensors to dynamically regulate the related genes in the 3HP biosynthesis pathway, induce the expression of related genes through different types and concentrations of inducers, negatively regulate the production of products in the metabolic pathway, reduce cytotoxicity and improve the ability of bacteria to synthesize 3HP. Background technique [0002] 3HP is an important platform chemical, ranking third among the twelve platform compounds obtained from biomass released by the US Department of Energy (DOE). In chemical terms, 3HP is a structural isomer of a three-carbon achiral optical organic compound and lactic acid, also known as hydroacrylic acid or ethylene lactic acid. Since 3HP contains hydroxyl and carboxyl difunctional groups, it can be used in the production of chemicals ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0008C12N9/93C12P7/42C12Y102/01075C12Y604/01002C07K14/32
Inventor 刘长莉隋广超徐启江杨洪一张杰常乐
Owner NORTHEAST FORESTRY UNIVERSITY
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