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Construction method of genetically engineered bacterium for producing astaxanthin

A technology of genetically engineered bacteria and astaxanthin, applied in the field of microbial fermentation, can solve the problems of cumbersome operation and long cycle, and achieve the effects of avoiding cloning, shortening construction time, and high efficiency of homologous recombination

Inactive Publication Date: 2014-06-18
SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method for constructing astaxanthin genetically engineered bacteria needs to sequentially integrate the genes related to astaxanthin expression on the yeast chromosome, involves multi-level cloning, and depends on the selection of suitable enzyme cutting sites. The operation is cumbersome and the cycle time long

Method used

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  • Construction method of genetically engineered bacterium for producing astaxanthin
  • Construction method of genetically engineered bacterium for producing astaxanthin
  • Construction method of genetically engineered bacterium for producing astaxanthin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 , experimental protocol and primer design

[0032] Experimental scheme design of the present invention is as follows:

[0033] Five genes crtE (GenBank: DQ016502. 1, derived from Xanthophyllomyces dendrorhous ATCC24202), crtYB (GenBank: AY177204.1, derived from Xanthophyllomyces dendrorhous ATCC24202), crtI (GenBank: AY177424.1, derived from Xanthophyllomyces dendrorhous ATCC24202), 3crt8:ABGenBrankevZ ( sp.SD212) and crtW (GenBank: CP001037.1, derived from Nostoc punctiforme PCC73102), and simultaneously expressed the ble gene (Zeocin resistance gene) as a screening marker, and selected the yeast chromosome δ site as the integration site, and each gene module was in The integrated assembly sequence on the chromosome is as follows figure 1 shown. The promoters and genes are numbered respectively, as shown in Table 1.

[0034] Table 1. Promoters and gene numbers

[0035] promoter number

promoter name

gene number

gene name

A ...

Embodiment 2

[0045] Example 2 , Construction of genetically engineered bacteria

[0046] 2.1 Construction of plasmids pYES2-CrtE, pYES2-CrtYB, pYES2-CrtI, pYES2-CrtZ and pSH62-CrtW

[0047] References (Ken Ukibe, Keisuke Hashida, Nobuyuki Yoshida, and Hiroshi Takagi. Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2009: 7205-7211; Venjnanger, JM, R .High-level production of Beta-carotene in Saccharomyces cerevisiae by successful transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol, 2007, 73:4342-4350) method, obtain crtE, crtYB and crtI genes, and insert them into pYES2 respectively .0 (purchased from Invitrogen) to obtain plasmids pYES2-CrtE, pYES2-CrtYB and pYES2-CrtI, transform them into E.coli strain DH5α, and save the plasmids.

[0048] A synthetic company (Nanjing GenScript Biotechnology Co., Ltd.) was commissioned to construct plasmids...

Embodiment 3

[0076] Example 3 , Fermentation of genetically engineered bacteria and detection of astaxanthin

[0077] 3.1. Fermentation

[0078] The seed cultivation method is as follows:

[0079] Pick a single colony of the positive clone obtained from the screening in Example 2 from the culture plate, insert it into 25ml of YPD medium (250ml shake flask), culture at 30°C, 220rpm / min, for 48h.

[0080] The fermentation culture method is as follows:

[0081]The seed solution was added to 25ml YPD medium (250ml shake flask) according to the inoculation ratio of 4%, cultured at 30°C and 220rpm / min for 72h.

[0082] 3.2. Extraction of Astaxanthin

[0083] The extraction method of astaxanthin is as follows:

[0084] Transfer the fermentation broth to a weighed centrifuge tube, centrifuge at 4°C, 4000r / min, for 5min, discard the supernatant, wash once with sterile water, discard the supernatant, and weigh. For each gram of wet bacteria, add 17.5ml of DMSO preheated to 55°C, mix well, and...

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Abstract

The invention discloses a construction method of genetically engineered bacterium for producing astaxanthin, which is characterized by comprising the following steps: A)constructing a gene expression module, wherein the gene expression module comprises relative gene for producing astaxanthin, a promoter at the upstream of relative gene for producing astaxanthin and a terminator at the downstream of the relative gene for producing astaxanthin; and B)performing cotransformation of the gene expression module to saccharomyces cerevisiae. The method has the advantages of simple, rapid and high efficiency performance, multi-grade clone is avoided, restriction enzyme cutting site is not required for depending on, multiple fragments enable cotransformation, the homologous recombination efficiency is high, and the engineering bacteria construction time can be obviously shortened.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a method for constructing astaxanthin-producing genetically engineered bacteria. Background technique [0002] So far, more than 600 carotenoids have been reported from both bacteria, algae, yeast and plants, and astaxanthin is one of them. The full chemical name of astaxanthin is 3,3′-dihydroxy-4,4′-diketone-β,β’-carotene, and its structural formula is: [0003] [0004] Astaxanthin, as the most effective natural coloring agent, is widely used in food and textile industries. In addition, astaxanthin is also an extremely effective antioxidant substance, which can protect cells from free radical damage. Due to its important functions of anti-cancer, enhancing the body's immunity and anti-aging, it is widely used in medicine, health care and cosmetics. Industry concerns. [0005] At present, the methods for producing astaxanthin mainly include chemical synthe...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/11C12R1/865
Inventor 朱丽蒋宇高书良夏兴杨晟戈梅
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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