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Dynamic regulation and control system for coumaric acid response and construction method thereof

A p-coumaric acid, dynamic response technology, applied in the field of metabolic engineering, can solve the problem of insufficient energy supply of bacteria, and achieve the effect of strengthening the synthesis of phenylpropanoid compounds and achieving balance

Active Publication Date: 2020-04-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Excessive consumption of acetyl-CoA to synthesize malonyl-CoA may lead to problems such as insufficient energy supply of bacteria

Method used

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  • Dynamic regulation and control system for coumaric acid response and construction method thereof
  • Dynamic regulation and control system for coumaric acid response and construction method thereof
  • Dynamic regulation and control system for coumaric acid response and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1P PadC Promoter structure simplification and biosensor functional verification

[0042] Due to the recognition of the coumaric acid response protein PadR the promoter P PadC The minimum structure of P PadC Promoter. Therefore, using a promoter truncation strategy, from P PadC The promoters are truncated to different degrees at both ends, and the truncated P PadC Express green fluorescent protein, clone into the BamHI / SpeI site of pRSM-T7-PadR plasmid, obtain a series of pRSM-T7-PadR-PpadC(P-Q)-EGFP plasmids ( figure 1 A). The plasmid is induced by IPTG to express PadR protein, thereby repressing the expression of downstream fluorescent proteins, and p-coumaric acid can bind to PadR protein to relieve the repression effect and turn on the expression of fluorescent proteins. First culture in LB medium at 37°C overnight, then inoculate into new LB medium with 1% inoculum the next day and cultivate to OD at 30°C 600 = 0.4-0.6, then add 0.01mM concentration o...

Embodiment 2

[0043] Example 2 Malonyl-CoA synthesis pathway optimization

[0044] On the basis of Example 1, using the simplified structure of P PadC Promoter constructs such as figure 2 Regulatory system shown. where P PadC (608-496) promoter regulates antisense RNA expression of gltA gene, P PadC The (608-476) promoter overexpresses the acs and ACC genes and expresses the above genes in combination. Seven pathways were optimally combined (including pRSM-PadR-acs, pRSM-PadR-PTasgltA, pRSM-PadR-acs-PTasgltA, pRSM-PadR-ACC, pRSM-PadR-acs-ACC, pRSM-PadR-PTasgltA-ACC and pRSM-PadR-acs-PTasgltA-ACC plasmid) was transformed into the naringenin-synthesizing strain. Firstly, three single clones of all engineering strains were picked as biological parallels, cultured in LB medium at 37°C overnight, and inoculated into fresh MOPS medium at 30°C, 220rpm for 48 hours the next day with an inoculum of 1%. The intracellular production of naringenin was then detected.

[0045] The results show th...

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Abstract

The invention discloses a dynamic regulation and control system for coumaric acid response and a construction method thereof, which belong to the technical field of metabolic engineering. On the basisof p-coumaric acid responsive protein PadR, the dynamic regulation and control system capable of sensing intracellular p-coumaric acid content change is established by utilizing regulation and control means such as a gene overexpression technology and an antisense RNA inhibition technology and combining with a naringenin anabolism pathway. According to the dynamic regulation and control system, malonyl-CoA synthesis can be enhanced when p-coumaric acid is excessive, and a downstream metabolic pathway is accelerated, so that the intracellular p-coumaric acid accumulation amount is reduced, naringenin synthesis is promoted, and the yield can be increased to 1.5 times of the original yield. According to the invention, the balance between cell growth and product synthesis is realized, and anidea is provided for metabolic engineering transformation of other high-added-value compound production strains.

Description

technical field [0001] The invention relates to a dynamic control system for responding to coumaric acid and a construction method thereof, belonging to the technical field of metabolic engineering. Background technique [0002] P-coumaric acid is the direct precursor of the synthesis of phenylpropanoid compounds, and it also has a variety of physiological functions, such as liver and gallbladder, heart protection, blood fat reduction, anti-cancer and anti-oxidation, etc., has been used in the fields of food and medicine is widely used. The supply of malonyl-CoA is often involved in the synthesis of phenylpropanoids (such as flavonoids and stilbenes) by microorganisms using Escherichia coli as the chassis. Insufficient supply of intracellular malonyl-CoA was an important reason for the lower final yield and accumulation of p-coumaric acid. Therefore, enhancing malonyl-CoA synthesis in microorganisms can help reduce the accumulation of p-coumaric acid and increase the produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/53C12N15/54C12N15/52C12N1/21C12R1/19
CPCC12N9/0006C12N9/1217C12N9/93C12N15/52C12N15/70C12N2830/001C12Y101/01C12Y207/02001C12Y604/01002
Inventor 周景文陈坚周胜虎邓禹堵国成
Owner JIANGNAN UNIV