Recombinant bacterium for producing beta-carotene as well as construction method and application of recombinant bacterium

A carotene and construction method technology, applied in the fields of metabolic engineering and combinatorial biology, can solve the problems of low stability and low yield, and achieve the effect of high-yield beta-carotene performance

Active Publication Date: 2017-07-28
SHAANXI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] The technical problem to be solved by the present invention is to provide a recombinant bacterium with high production of β-carotene and a method for constructing the recombinant bacterium aiming at the problems of low stability and low yield in the existing methods for producing β-carotene and application

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  • Recombinant bacterium for producing beta-carotene as well as construction method and application of recombinant bacterium

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Embodiment 1

[0030] according to figure 1 The β-carotene biosynthetic pathway in Yarrowia lipolytica is shown, and the recombinant bacterium producing β-carotene is constructed. The specific construction method is as follows:

[0031] 1. Construction of gene expression module

[0032]The codon-optimized phytoene synthase / lycopene cyclase gene (opt carRA, whose base sequence is shown in SEQ ID No.1 and whose accession number is KY971027, was provided by Nanjing GenScript Biotechnology Co., Ltd. Synthesized by the company) and expression vector pJN44 (containing promoter P TEF and terminator T xpr2 ) were treated with restriction endonuclease SmaI at 37°C for 2 h, purified and gel-recovered the DNA fragments of the target gene and the vector; the two recovered DNA fragments were treated with NEB DNA ligase (NEB Company, product number: M0367S) The ligation reaction was carried out to obtain the plasmid optpJN44-carRA containing the expression module of optcarRA.

[0033] The codon-optimi...

Embodiment 2

[0066] The application of the recombinant bacteria of embodiment 1 in the preparation of β-carotene, the specific method is as follows:

[0067] 1. Primary seed culture: inoculate the recombinant bacterial strain of Example 1 into a test tube containing 5 mL of seed culture solution, and cultivate it at 28°C and 200 rpm / min for 24 hours to obtain a first-class seed solution; wherein the seed culture solution is Add 2g of glucose, 2g of peptone, and 1g of yeast powder per 100mL of deionized water, and obtain it after sterilization.

[0068] 2. Fermentation culture: Transfer the primary seed solution to a 250mL shake flask containing 50mL of fermentation medium at an inoculum size of 1%, culture it at 28°C and 200rpm / min for 144h, and collect the bacterial solution; It is obtained by adding 4g of glucose, 0.251g of ammonium sulfate, 0.2g of yeast nitrogen base lacking leucine (Drop-out mix syntheticminus leucine w / o yeast nitrogenbase), and 0.17g of yeast nitrogen base per 100mL...

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Abstract

The invention discloses a recombinant bacterium for producing beta-carotene as well as a construction method and application of the recombinant bacterium. The construction method comprises the following steps: respectively constructing superstrong gene expression modules of protein phytoene synthetase / lycopene cyclase coded by beta-carotene synthetic genes optimized by codons, phytoene dehydrogenase, protein geranyl diphosphate synthase coded by MAV (micro aerial vehicle) pathway genes and 3-hydroxy-3-methylglutaryl coenzyme A reductase; assembling the gene expression modules in vitro, so as to obtain four plasmids of the gene expression modules, integrating the plasmids into a host bacteria genome which does not produce beta-carotene and screening orange single colonies, so as to obtain the recombinant bacterium for producing beta-carotene. The recombinant bacterium disclosed by the invention is adopted for producing beta-carotene by fermental cultivation, and the yield of beta-carotene can reach 26.03mg / g DCW. Therefore, the recombinant bacterium has high performance of producing beta-carotene.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering and combined biology, and in particular relates to a recombinant bacterium producing beta-carotene and its construction method and application. Background technique [0002] Beta-carotene is a natural carotenoid found in many foods such as carrots, papayas, and green vegetables. In nature, β-carotene mainly exists in the all-trans structure, has 11 conjugated carbon-carbon double bonds, has good effects of quenching free radicals, anti-oxidation, and inhibiting singlet oxygen, and has anti-cancer, anti- Cancer, anti-aging and other functions. As the precursor of vitamin A, β-carotene is a type A nutritional food fortifier recognized by the WHO and FAO Joint Expert Committee on Food Additives. As a drug, β-carotene has been officially recorded in the 1990 and 1995 editions of the United States Pharmacopoeia (USP), the 1997 edition of the European Pharmacopoeia, and the 1998 edition ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/19C12P23/00C12R1/645C12R1/865C12R1/72C12R1/19
CPCC12N9/0006C12N9/001C12N9/1085C12P23/00C12Y101/01088C12Y103/99031C12Y205/01029
Inventor 屈玉玲孟永宏
Owner SHAANXI NORMAL UNIV
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