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Construction and application of astaxanthin synthesis strain

A technology of astaxanthin and recombinant bacteria, applied in the directions of enzymes, bacteria, isomerases, etc., can solve the problems that the protein cannot be folded correctly, limit the production of astaxanthin, and it is difficult to produce high yields, so as to eliminate the toxicity of metabolic intermediates, increase the Astaxanthin production, the effect of increasing the production capacity

Active Publication Date: 2021-05-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to factors such as long metabolic pathway of astaxanthin, toxicity of metabolic intermediates, and product toxicity, it is difficult to achieve high yield
In addition, β-carotene hydroxylase (crtZ) and β-carotene ketoylase (crtW) have membrane protein properties, which may cause the protein to fail to fold correctly and affect protein activity, thereby limiting the production of astaxanthin

Method used

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  • Construction and application of astaxanthin synthesis strain
  • Construction and application of astaxanthin synthesis strain
  • Construction and application of astaxanthin synthesis strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, construction of astaxanthin-producing bacterial strain ast005

[0065] (1) Construction of β-carotene producing strain CAR026

[0066] Lycopene β-cyclase (CrtY, encoded by crtY) from Pantoea agglomerans can catalyze the synthesis of β-carotene from lycopene. In this example, the crtY gene was linked to the M1-93 promoter and integrated into the pta site of the LYC102 strain by a two-step regulation method to construct a high-yielding β-carotene strain CAR026. Specific steps are as follows:

[0067] 1. Escherichia coli LYC102 with pKD46

[0068] The plasmid pKD46 was transformed into Escherichia coli LYC102 to obtain Escherichia coli LYC102 carrying pKD46.

[0069] 2. Construction of CAR026 strain by two-step homologous recombination

[0070] 1) Step 1 Homologous recombination construction of intermediate bacteria expressing cat-sacB

[0071] Using the pXZ-CS plasmid as a template, pta-cat-up and pta-cat-down as primers, PCR obtained about 3000bp DNA f...

Embodiment 2

[0129] Embodiment 2, the construction of producing astaxanthin bacterial strain

[0130] A second copy of the crtZ gene was added to the astaxanthin-producing recombinant strain Ast005 prepared in Example 1 to reduce the accumulation of canthaxanthin and β-carotene and increase the synthesis of astaxanthin.

[0131] 1. Astaxanthin-producing Ast006 and the acquisition of library strains

[0132] 1. One-step homologous recombination method to add a second copy of the crtZ gene

[0133] The pKD46 plasmid was transformed into Escherichia coli ast005 prepared in Example 1 to obtain Escherichia coli Ast005 carrying pKD46.

[0134] Using pflB-up-FRT / M1P-R as primers and using the genome of the recombinant strain M1-93 as a template, amplify about 1600bp DNA fragment I;

[0135] Using crtZ-M1-F / pflB-crtZ-R as primers, using Pantoea agglomerans (Pantoea agglomerans, purchased from the Institute of Microbiology, Chinese Academy of Sciences, product number: 1.2244 genomic DNA as a temp...

Embodiment 3

[0154] Example 3, Construction of astaxanthin-producing strains expressing molecular chaperone genes

[0155] 1. Molecular chaperone gene regulation increases astaxanthin production

[0156] The two-step homologous recombination regulates the molecular chaperone genes groES and groEL in Ast007; the molecular chaperones GroES and GroEL can help foreign proteins to fold correctly. Because the two genes are under the same operon, use the method of two-step homologous recombination to replace the groES-groEL gene (sequence 14, wherein the 1-294bp is the groES sequence, The 338-1984bp is the groEL sequence, and the 294-337bp sequence is the sequence between the two genes) the front part of the operon (the front part of the gene) to achieve the purpose of regulating the molecular chaperone genes groES and groEL, and improve the activity of CrtZ and CrtW (through Increase the correct folding of CrtZ and CrtW proteins, increase the amount of active proteins, thereby increasing protei...

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Abstract

The invention discloses construction and an application of an astaxanthin synthesis strain. The invention provides a recombinant bacterium, which is prepared according to a method comprising the following steps of: expressing and synthesizing carotenoid related genes crtY gene, crtZ gene and crtW gene in escherichia coli, and regulating and controlling the expression of molecular chaperone genes groES and groEL in the escherichia coli to obtain the recombinant bacterium, according to the invention, the promoters with different intensities are utilized and gene copy is changed, so that coordinated expression among genes in a synthetic pathway of astaxanthin is realized, and the toxicity of metabolic intermediates is eliminated, so that the generation capacity of biosynthesis of astaxanthin is improved. In addition, stabilizing the expression of the exogenous gene and increasing the correct folding of the exogenous gene can also become an effective way for improving the biological activity of astaxanthin synthetic pathway enzyme and increasing the yield of astaxanthin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of astaxanthin-synthesizing bacterial strains. Background technique [0002] Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-diketonyl) is a high-value ketocarotenoid with strong antioxidant and mono Heavy oxygen quenching activity can regulate biological functions ranging from lipid peroxidation to tissue resistance to UV damage. Of the more than 600 known natural carotenoids, beta-carotene, lutein, lycopene, zeaxanthin, and astaxanthin, the most powerful antioxidant known , which is about 100 times more potent than other carotenoids such as beta-carotene, lutein and vitamin E. Thus, astaxanthin is able to protect cells, lipids and membrane lipoproteins from oxidative damage. In addition, astaxanthin has a variety of other biological properties, including anti-inflammatory, anti-cancer, and immune system-boosting properties. As an appr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P23/00C12R1/19
CPCC12N15/52C12N9/90C12Y505/01019C12N9/0077C12N9/0083C12N15/70C12P23/00
Inventor 张学礼李清艳唐金磊龚忠阔秦莹刘茹
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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