Genetically engineered bacterium for improving yield of beta-carotene and application thereof

A carotene and gene technology, applied in the direction of enzymes, fungi, acyltransferases, etc., can solve the problems of not being able to further break through the yield, not considering the use of carbon sources, and reducing power supply for the storage of final products, so as to increase the content of lipid droplets and Volume, reduce carbon loss, increase storage effect

Active Publication Date: 2021-07-23
GUANGZHOU WISDOM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many reports on the synthesis of β-carotene by genetically modified microbial engineering bacteria, these modifications only strengthen the gene expression of the metabolic pathway from acetyl-CoA to β-carotene through mevalonate, without considering the carbon source Utilization, supply of reducing power, and storage of final products are such short-board problems that it is impossible to further break through the output to meet the requirements of industrial production

Method used

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  • Genetically engineered bacterium for improving yield of beta-carotene and application thereof
  • Genetically engineered bacterium for improving yield of beta-carotene and application thereof
  • Genetically engineered bacterium for improving yield of beta-carotene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1 gene selection and vector construction

[0112] Acetyl-CoA is an important precursor for the synthesis of fatty acids, organic acids and terpenes by microorganisms, but the natural synthesis of acetyl-CoA in most microbial strains results in the loss of carbon. The phosphoketolase pathway (eg figure 1 Shown) phosphoketolase (PK) and phosphate acetyltransferase (PTA) can catalyze 6-phosphate fructose (F6P) or 5-phosphate xylulose (X5P) to generate acetyl-CoA, the process does not generate carbon dioxide, no carbon It also produces ATP and NADPH to provide energy and reducing power for cellular anabolism.

[0113] Lipid droplets (LD) are organelles widely present in cells, which are surrounded by neutral lipids with a single layer of phospholipid membrane. Lipid droplets generate malonyl-CoA under the catalysis of ACC1 through acetyl-CoA, which generates palmitoyl-CoA and stearoyl-CoA under the action of FAS, and these two saturated fatty acids generate ole...

Embodiment 2

[0161] Example 2 Construction of high-yield β-carotene integrated strain

[0162] Recombinant engineering bacteria randomly integrate plasmid pZ1, plasmid pZ2, plasmid pZ3, plasmid pZ4, plasmid pZ5, and plasmid pZ6 into the genome of Yarrowia lipolytica through non-homologous recombination; including the following steps:

[0163] (1) The constructed recombinant plasmid pZ1 was linearized by excising the original pUC19 backbone with NotⅠ, and then introduced into Yarrowia lipolytica strain Polf, which is deficient in uracil synthesis, and the strain with the highest β-carotene production was detected to complete the first round of strain recombination iterate.

[0164] (2) Excision of the URA3 gene by introducing a circular plasmid expressing Cre enzyme Y-Cre to restore the selection marker to the first-generation engineering strain producing β-carotene, and then re-introducing the linearized pZ2 plasmid and screening for high-yield β-carotene to complete the second stage Two ...

Embodiment 3

[0174] Embodiment 3 industrial fermentation test

[0175] After the genetically engineered bacteria ZTQ211, ZTQ30 and ZTQ5320 constructed in Example 2 were subjected to high-density fermentation in a 3L fermenter, the unit yield of β-carotene was detected.

[0176] The fermentation method comprises the following steps:

[0177] S1. Cultivation of primary seed liquid: inoculate a single colony of the recombinant strain F5 on a solid YPD plate into a 100ml Erlenmeyer flask containing 20ml of YPD liquid medium, and cultivate for 48 hours at 30°C with a shaker speed of 200rpm.

[0178] S2. the primary seed solution obtained in step S1 is transferred to the 300ml Erlenmeyer flask containing 100ml YPD liquid medium by 1% (wt) inoculum, 30 ℃, shaker speed 200rpm, cultivate 10 hours cell OD600 reaches 10 , to obtain the secondary seed solution.

[0179] S3. The secondary seed liquid obtained in step S2 is transferred to the 3L fermenter containing 1L fermentation medium by 10% (wt) ...

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Abstract

The invention discloses a genetically engineered bacterium capable of increasing the yield of beta-carotene and application thereof, and the genetically engineered bacterium contains a beta-carotene synthetic pathway related gene, a phosphoketolase pathway related gene and a lipid droplet synthetic pathway related gene. A beta-carotene synthetic pathway is covered; a phosphoketolase pathway is increased, the carbon loss is reduced, and more precursor substance acetyl coenzyme A for synthesizing beta-carotene is generated from a carbon source; the supply of a reducing power substance NADPH and an energy substance ATP is increased; and a lipid droplet synthesis pathway is also increased, and the storage of beta-carotene can be increased by increasing the content and volume of intracellular lipid droplets. The highest yield of the beta-carotene of the genetically engineered bacterium reaches 7.8g / L, and the yield of the beta-carotene is remarkably improved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a genetically engineered bacterium capable of increasing the biosynthetic output of β-carotene, its construction method and application. Background technique [0002] β-carotene is a tetraterpene compound composed of 8 isoprene units connected by unsaturated carbon bonds. It has superior physiological functions and is widely used in pharmaceutical and health products, food additives, cosmetics and feed additives industries. . [0003] The preparation of β-carotene mainly relies on chemical synthesis. Unlike natural β-carotene, which is a mixture of trans and cis configurations, chemically synthesized β-carotene is almost all in trans configuration, which greatly reduces the absorption rate of β-carotene by the human body. In addition, the chemical synthesis method has complicated process, high energy consumption and heavy pollution. The microbia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P23/00C12R1/645
CPCC12N15/815C12P23/00C12Y604/01002C12Y203/0102C12Y114/19001C07K14/39C12N9/1029C12Y203/0301C12Y101/01088C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y205/01001C12Y205/01029C12Y502/01013C12Y103/99031C12Y205/01032C12Y401/02022C12Y401/02009C12N9/93C12N9/0071C12N9/1025C12N9/0006C12N9/1205C12N9/1229C12N9/88C12N9/90C12N9/1085C12N9/001
Inventor 吴世林李强祝重阳江华峰
Owner GUANGZHOU WISDOM BIO TECH
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