General chassis cell for synthesizing acetylcoenzyme A derived product and construction method and application of general chassis cell

A technology of acetyl coenzyme and derivative products, applied in the field of genetic engineering, can solve the problems such as cumbersome transformation process of recombinant bacteria, lack of versatility, time-consuming, etc., achieves great application potential, reduces the synthesis of acetic acid by-products, and has a wide range of applications. Effect

Active Publication Date: 2020-01-07
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that the existing recombinant bacteria are only suitable for the biosynthesis of a specific single product and do not have versatility, if the transformation process of the recombinant bacteria is cumberso

Method used

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  • General chassis cell for synthesizing acetylcoenzyme A derived product and construction method and application of general chassis cell
  • General chassis cell for synthesizing acetylcoenzyme A derived product and construction method and application of general chassis cell
  • General chassis cell for synthesizing acetylcoenzyme A derived product and construction method and application of general chassis cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: the construction of universal chassis cell

[0071] 1. Gene knockout

[0072] Using the P1 phage transduction method (principle such as figure 1 ) to knock out the global regulatory factor arcA and the acetate kinase gene ackA of Escherichia coli E. coli BL21 (DE3) genome respectively.

[0073] 1. The knockout method of the global regulatory factor arcA:

[0074] 1) Phage activation

[0075] Add 4ml of heated and melted 0.4% agar medium to a 10ml sterile EP tube, add 400 μL of overnight cultured donor strain JW4364 (the donor strain is derived from the Keio Collection library, which can be purchased commercially, and the KeioCollection library construction method is as follows: Baba T, et al. Construction of Escherichia coli K-12in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006, 2(1): 1-11), add 10 μL of stored phage stock solution, and mix the solution Then pour it on the LB plate without anti-antibody, and cu...

Embodiment 2

[0097] Example 2 Biosynthesis of Acetyl-CoA Derivatives

[0098] This embodiment lists the synthesis experiments of two important acetyl-CoA derivatives, phloroglucinol (PG) and 3-hydroxypropionic acid (3HP), to illustrate the general application of the chassis cells of the present invention in the biosynthesis of acetyl-CoA derivatives. sex.

[0099] 1. Detection of PG biosynthesis

[0100] 1. Construction of PG synthetic strains

[0101] Prepare the wild-type control strain E.coliBL21(DE3) and the acetyl-CoA derivative product general-purpose chassis cells (E.coli BL21(DE3)△arcA△ackAT7-csrB) constructed in Example 1 according to the operation steps of the TAKARA Competent Preparation Kit T7-acs), the recombinant plasmids pET-phlD-mar and pA-accADBC of the acetyl-CoA derivative product PG synthesis pathway (the construction method of the plasmid refers to Cao Y, et al.Improved phloroglucinol production by metabolically engineered Escherichia coli.Appl Microb Biotechnol 201...

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Abstract

The invention discloses a general chassis cell for synthesizing an acetylcoenzyme A derived product and a construction method and application of the general chassis cell, and belongs to the technicalfield of genetic engineering. The chassis cell adopts escherichia coli as a start bacterial strain, overall situation regulating factors arcA and acetate kinase genes ackA in escherichia coli genome are knocked out, and carbon storage regulatory factors csrB and acetylcoenzyme A synthetase genes acs are subjected to over expression. Besides, the invention further provides a preparation method of the chassis cell, and a method for producing the acetylcoenzyme A derived product by using the chassis cell. The invention firstly completes establishment of the general chassis cell through biosynthesis of the acetylcoenzyme A derived product, the purpose of reforming production is realized, and the method has great innovativeness and application potential. The chassis cell is suitable for biosynthesis of the acetylcoenzyme A derived product.

Description

technical field [0001] The invention relates to a general-purpose chassis cell for synthesizing acetyl-CoA derivatives, a construction method and application thereof, and belongs to the technical field of genetic engineering. Background technique [0002] Acetyl-CoA is a core metabolite in cells, which is essential for maintaining the normal life activities of cells. Acetyl-CoA can be used as a precursor to synthesize various types of derivative products, such as isoprene and terpenoids, polyketides, polyhydroxy fatty acids, lipids, etc., which have been widely used in medicine, food additives, chemicals, agriculture, cosmetics And other major fields. [0003] At present, there are many studies on the biosynthesis of acetyl-CoA derivatives at home and abroad, and many important research progresses have been made. For example, phloroglucinol (PG) is an intermediate in the synthesis of flavonoids and isoflavones, and has a wide range of anticancer and anticardiovascular effe...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/22C12P7/42C12R1/19
CPCC12N15/70C12P7/22C12P7/42
Inventor 赵广刘敏咸漠赵喆
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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