Construction method of genetically engineered bacterium for producing beta-carotene

A technology of genetically engineered bacteria and carotene, applied in the field of construction of genetically engineered bacteria producing β-carotene, can solve the problems of cumbersome operation and long cycle, and achieve the effects of avoiding cloning, shortening construction time, and good industrial application prospects

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 of constructing β-carotene genetically engineered bacteria needs to sequentially integrate the genes related to β-carotene expression into

Method used

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  • Construction method of genetically engineered bacterium for producing beta-carotene
  • Construction method of genetically engineered bacterium for producing beta-carotene
  • Construction method of genetically engineered bacterium for producing beta-carotene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 , experimental protocol and primer design

[0031] The experimental scheme design of this embodiment is as follows:

[0032] In Saccharomyces cerevisiae BY4742 (purchased from EUROSCARF, No. Y10000, strain information: MATα; his3△1; leu2△0; lys2△0; ura3△0) expressed the β-carotene synthesis pathway from Phaffia rhodozyma ATCC24202 Three genes crtE (GenBank: DQ016502.1), crtYB (GenBank: AY177204.1) and crtI (GenBank: AY177424.1), and simultaneously express the ble gene (Zeocin resistance gene) as screening markers, select the δ position of yeast chromosome point as the integration site, and the integration and assembly sequence of each gene module on the chromosome is as follows: figure 2 shown. The promoters and genes are numbered respectively, as shown in Table 1.

[0033] Table 1. Promoters and gene numbers

[0034] promoter number

promoter name

gene number

gene name

A

TEF1p

1

CrtE

B

TPI1p

2

w...

Embodiment 2

[0044] Example 2 , Construction of genetically engineered bacteria

[0045] 2.1. Construction of plasmids pYES2-CrtE, pYES2-CrtYB and pYES2-CrtI

[0046] References (Ken Ukibe, Keisuke Hashida, Nobuyuki Yoshida, and Hiroshi Takagi. Metabolic Engineering of Saccharomyces cerevisiae for AstaxanthinProduction and Oxidative Stress Tolerance. APPLIED AND ENVIRONMENTALMICROBIOLOGY, 2009: 7205-7211. level production of Beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol, 2007, 73:4342-4350) to obtain crtE, crtYB and crtI genes, and insert them into pYES2.0 (purchased From Invitrogen), the plasmids pYES2-CrtE, pYES2-CrtYB and pYES2-CrtI were obtained, transformed into E.coli strain DH5α, and the plasmids were preserved.

[0047] 2.2. Extraction of template DNA

[0048] The extraction method of Saccharomyces cerevisiae BY4742 chromosomal DNA is as follows: pick a plump single colo...

Embodiment 3

[0080] Example 3 , fermentation of genetically engineered bacteria and detection of β-carotene

[0081] 3.1. Fermentation

[0082] The seed cultivation method is as follows:

[0083] 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.

[0084] The fermentation culture method is as follows:

[0085] The seed liquid was added to 25ml YPD medium (250ml shake flask) according to the inoculation ratio of 4%, and cultured at 30°C and 220rpm / min for 120h.

[0086] 3.2. Extraction of β-carotene

[0087] The extraction method of β-carotene is as follows:

[0088] 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, an...

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Abstract

The invention discloses a construction method of genetically engineered bacterium for producing beta-carotene, which is characterized by comprising the following steps: A)constructing a gene expression module, wherein the gene expression module comprises relative gene for producing beta-carotene, a promoter at the upstream of relative gene for producing beta-carotene and a terminator at the downstream of the relative gene for producing beta-carotene; 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 construction method of a β-carotene-producing genetically engineered bacterium. Background technique [0002] So far, more than 600 carotenoids have been reported from bacteria, algae, yeast and plants, and β-carotene is one of them. As one of the precursors of vitamin A, β-carotene is a class A complete nutritional food fortifier identified by the Food and Agriculture Organization of the United Nations and the World Health Organization Joint Expert Committee on Food Additives. It has been approved for use by 52 countries and regions in the world, and Included in the Food Chemical Additives Code (FCC) of the United States. In addition, as a drug, β-carotene has been officially included in the 1990 and 1995 editions of the United States Pharmacopoeia (USP), the 1997 edition of the European Pharmacopoeia, and the 1998 edition of the British Pharmacopoeia (BP). ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/11C12R1/865
Inventor 蒋宇朱丽高书良罗敏玉戈梅杨晟
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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