Construction method of engineering bacteria capable of dynamically regulating synthesis of 3HP (3-hydroxypropionic acid)

A technology of hydroxypropionic acid and engineering bacteria, applied in the field of genetic engineering, can solve the problems of reducing product concentration, limiting the accumulation of downstream products, and low efficiency.

Pending Publication Date: 2017-11-28
NORTHEAST FORESTRY UNIVERSITY
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Problems solved by technology

[0004] In our previous research on 3HP synthesis, we found that the conversion of acetate CoA to malonyl CoA was inefficient, which limited the accumulation of downstream products.
In order to improve the efficiency of malonyl-CoA synthesis, acc was overexpressed, but as the concentration of the inducer IPTG increased, the concentration of the final product was found to gradually decrease
In order to explore the reason for its decrease, we respectively used the acc gene from E. coli and Saccharomyces cerevisiae (acc E and acc S ) and the pE7a plasmid without the acc gene were expressed in E.coli respectively, and it was found that although the high concentration of IPTG had a certain effect on the concentration of the control cells that did not synthesize malonyl CoA, compared with the control, acc E and acc S The expression of the expression of the host bacteria significantly inhibited the growth of

Method used

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  • Construction method of engineering bacteria capable of dynamically regulating synthesis of 3HP (3-hydroxypropionic acid)
  • Construction method of engineering bacteria capable of dynamically regulating synthesis of 3HP (3-hydroxypropionic acid)
  • Construction method of engineering bacteria capable of dynamically regulating synthesis of 3HP (3-hydroxypropionic acid)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of Overexpression Acetyl CoA Carboxylase Plasmid pE7a-acc

[0028] In E. coli, malonyl-CoA is biosynthesized by acetyl-CoA by the acetyl-CoA carboxylase encoded by accABCD (acc). The acetyl-CoA carboxylase of Escherichia coli consists of four subunits: biotin carboxyl carrier protein (accB), biotin carboxylase (accC) and two carboxyltransferase subunits (accD, accA). Genomic DNA was extracted from Escherichia coli with a bacterial genome extraction kit purchased from Tiangen Company, and PCR amplification of the target DNA fragment was carried out with primers designed by the biological company. Amplification primers: accD-f: 5'-CGCGGATCCGATGAGCTGGATTGAACGAATTAAAAG-3', accD-r: 5'-ATGCGAGCTCTCAGGCCTCAGGTTCCTGATCPCR-3'; accA-f: 5'-CGCGGATCCGATGAGTCTGAATTTCCTTGATTTTG-3', accA-r: 5'-ATGCGAGCTCTTACGC-GTAACCGTAGCTCA '; accBC-f: 5'-GGAATTCCATATGGAT ATTCGTAAGATTAAAAAAC-3', accBC-r: 5'-CCGCTCGAGTTATTTTTTCCTGAAGACCGAG-3'. Amplification system (100μl): DD...

Embodiment 2

[0029] Example 2: Construction of expressing malonyl-CoA reductase plasmid pS2c-mcr

[0030]The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the two-step NADPH-dependent reduction of malonyl-CoA to produce 3HP. Chloroflexus aurantiacus genomic DNA was extracted with a bacterial genome extraction kit purchased from Tiangen, and the designed primers were sent to the company for synthesis: mcr-f: 5'-TT TTTGAATTCATGAGCGGAACAGGACGAC-3', mcr-r: 5'-TTTTTGGATCCTTA CACGGTAATCGCCCGTC-3' for amplification of the mcr gene. At the same time, the plasmid backbone pS2c was amplified, and its primers were pS2c-f: 5'-TTTTTGGTCTCCAATTCTTACACGGTAATCGCCCGTC-3', pS2c-r: 5'-TTTTTGGTCTCAATGTATATCTCTTCTTAAAAGATC-3'. Amplification system (100μl): DDW 49μl+Betaine 20μl+DMSO 3μl, 5×Long Taq reaction buffer (Mg + plus) 20 μl, Primer mixed 5 μl, Template 1 μl, Phusion polymerase 0.6 μl. PCR amplification conditions: after pre-denaturation at 97°C for 5 minutes, denaturation at 94°C...

Embodiment 3

[0031] Embodiment 3: Containing the construction of biosensor controller plasmid pBFR1k-lacI-8FapR

[0032] The malonyl-CoA sensing controller is divided into two parts: one is the FapR sensor: the transcription factor FapR capable of responding to malonyl-CoA is cloned from Bacillus subtilis, and the P BAD The 3' of the promoter produces pA8c-FapR; the second is the promoter controller that regulates FapR (P FR1 ): Design and regulate the promoter sequence of FapR, that is, P FR1 . Amplification of the 17 bp DNA sequence naturally occurring in Bacillus subtilis that binds to FapR for insertion into P A1 Between the promoter -35 / -10, replace the original P A1 Promoter sequence, construction of promoter sequence P FR1 . The plasmid pBFR1k-lacI was obtained by amplifying the lacI gene from Escherichia coli, and then the P of pA8c-FapR BAD -FapR operon amplified and inserted into pBFR1k-la cI P FR1 The 5' end of the -lacI operon constitutes the final plasmid pBFR1k-lacI-8F...

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Abstract

The invention relates to a construction method of engineering bacteria capable of dynamically regulating synthesis of 3HP (3-hydroxypropionic acid). The construction method is characterized in that a transcription factor FapR based on bacillus subtilis has the characteristic of response to malonyl CoA, an acc gene synthesizing malonyl CoA and a mcr gene converting malonyl CoA into 3HP in a 3HP synthesis pathway are arranged in the downstream position of a promoter, transcription of the two genes is dynamically regulated according to the concentration of malonyl CoA, the engineering bacteria synthesized from 3HP are dynamically regulated according to the concentration of malonyl CoA, the problems that malonyl CoA poisons bacteria and the 3HP synthesis efficiency is low at present are solved, and the technical support is provided for mass production of 3HP.

Description

technical field [0001] The invention designs a construction method for engineering bacteria that dynamically regulates the synthesis of 3-hydroxypropionic acid (3-Hydroxypropionic acid, 3HP), and specifically relates to the technical field of genetic engineering. Background technique [0002] With the depletion of oil reserves and the deterioration of the environment, countries all over the world are devoting themselves to the research and development of various renewable energy sources. 3HP is one of the 12 value-added platform compounds in renewable biomass proposed by the US Department of Energy. The 3HP molecule contains two functional groups with different properties - hydroxyl and carboxyl, making it a precursor of many optically active substances, such as acrylic acid, 1,3-propanediol, 3-hydroxypropionaldehyde and malonic acid, etc. The application of this compound in the chemical industry is also diversified, it is used as polymer coating, metal lubricant and textil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
CPCC12N9/0006C12N9/93C12N15/70C12Y604/01002C07K14/32
Inventor 刘长莉李玉花徐启江刘曦王宏伟常乐
Owner NORTHEAST FORESTRY UNIVERSITY
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