Method of producing beta-carotene by genetically engineered bacterium and genetically engineered bacterium thereof

A technology of genetically engineered bacteria and carotene, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as high cost, complicated fermentation conditions, and difficult industrial production, and achieve high yield and simple fermentation conditions , significant production value effects

Inactive Publication Date: 2018-06-05
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of algae and yeast is relatively low and cannot meet the requirements of industrial production
[0005] Blakeslea trispora is currently the preferred strain for producing β-carotene, but the culture medium for producing β-carotene by Blakeslea trispora is complex and relatively expensive
In industrial production, the ratio of "+" and "-" strains is often considered, and the fermentation conditions are relatively complicated, which also brings many unnecessary difficulties to industrial production.

Method used

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  • Method of producing beta-carotene by genetically engineered bacterium and genetically engineered bacterium thereof
  • Method of producing beta-carotene by genetically engineered bacterium and genetically engineered bacterium thereof
  • Method of producing beta-carotene by genetically engineered bacterium and genetically engineered bacterium thereof

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Experimental program
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Effect test

Embodiment 1

[0058] The import of embodiment 1 Yarrowia lipolytic yeast gene

[0059] This example mainly describes the gene transformation method of Yarrowia lipolytica.

[0060] Competent cell preparation process

[0061] 1. Inoculate a single colony of Yarrowia lipolytica into 5ml of YPD liquid medium (citric acid-sodium citrate buffer to adjust the pH to 4.0), 28°C, 220rpm shaking culture for 10-12h;

[0062] 2. Transfer 50-100 μL of the above culture solution to 10 ml YPD liquid medium (pH 4.0), and culture overnight at 28°C and 220 rpm until the cell OD 600 About 5.5 or so;

[0063] 3. Centrifuge the culture medium at 28°C and 3500rpm for 5min;

[0064] 4. Discard the supernatant and wash the cells twice with 10ml 1×TE buffer;

[0065] 5. Dilute the suspension with about 20ml of 0.1M LiAc (pH6.0) to make the cell concentration reach 5×10 7 / ml;

[0066] 6. Gently incubate the suspension at 28°C, 100rpm for 1h;

[0067] 7. Centrifuge the culture solution at 28°C and 3500rpm for...

Embodiment 2

[0073] The acquisition of embodiment 2 URA3 deletion strain

[0074] HMG-CoA reductase is an important rate-limiting enzyme in the mevalonate pathway, catalyzing the conversion of HMG-CoA to mevalonate. To increase β-carotene production, we redirected carbon metabolism to the isoprenoid pathway by cloning expressing the catalytic site of HMG-CoA reductase. The HMG-CoA reductase gene from Yarrowia lipolytica was cloned, totaling 3000bp, its catalytic site was intercepted at 1500-3000bp, and the ATG start codon was added at the start site. In order to make this sequence express normally, the Exp1 promoter and the Aco terminator were added upstream and downstream respectively to obtain the complete cloned expression element Exp1-HMG-CoA cata -Aco, and then integrate the upstream and downstream homologous fragments of the URA3 gene to obtain the plasmid pSK-HMG cata -URA3.5N-3N, the sequence of this plasmid is shown in SEQ ID NO1 (referring to table 2, figure 2 ). The PmeI / Sp...

Embodiment 3 3

[0077] Embodiment 3 Expression of B. trispora β-carotene gene

[0078] This embodiment mainly describes the construction and expression of B. trispora β-carotene in Yarrowia lipolytica strain (see figure 1 ).

[0079]The constructed integrated plasmid pS-B-RA-GGS-F-U(L) mainly includes carB (lycopene dehydrogenase) and carRA (lycopene synthase and lycopene cyclase) derived from B. trispora ) and the GGS gene (bisgeranyl pyrophosphate synthase).

[0080] The GGS gene is cloned from Yarrowia lipolytica ATCC20362, and the specific sequence of the GGS gene is shown in SEQ ID NO.2.

[0081] The CarRA gene was cloned from B. trispora, and optimized according to the codon preference of Yarrowia lipolytica. The sequence of the optimized CarRA gene is shown in SEQ ID NO.3.

[0082] The CarB gene was cloned from B. trispora, and optimized according to the codon preference of Yarrowia lipolytica. The sequence of the optimized CarB gene is shown in SEQ ID NO.4. The above genes were ad...

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Abstract

The invention discloses a method of producing beta-carotene by a genetically engineered bacterium and the genetically engineered bacterium thereof. The genetically engineered bacterium is a yarrowia lipotica genetically engineered bacterium comprising a carRA gene and a carB gene. By introducing the carRA gene and the carB gene originated from blakeslea trispora into the yarrowia lipotica yeast bymeans of a genetic engineering method, expression of GGS and 3-hydroxyl-3-methyl acetyl coenzyme A reductase is further enhanced. 3-phosphoglycerol dehydrogenase (Gut2) and peroxidase (Pox2) are knocked out to obtain a yarrowia lipotica strain expressing beta-carotene heterologously. The method is simple in fermenting condition and relatively high in output of beta-carotene, and has important production value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing beta-carotene by a genetically engineered bacterium and the genetically engineered bacterium. Background technique [0002] β-carotene is an orange-yellow fat-soluble carotenoid, which is a type A nutritional food additive identified by the Food and Agriculture Organization of the United Nations and the World Health Organization Joint Expert Committee on Food Additives. In the human body, β-carotene is the precursor of vitamin A; it can act as an antioxidant to scavenge free radicals in the body, reduce the risk of heart failure in the elderly; stimulate the immune system, and has anti-cancer and anti-cancer functions. In addition, β-carotene can also be used in food pigments, animal feed additives, health products, cosmetics and pharmaceuticals. At present, the market demand for β-carotene continues to increase, and its application fields have also shifted fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P23/00C12R1/645
CPCC12N9/001C12N9/90C12P23/00C12Y103/99029C12Y505/01018C12Y505/01019
Inventor 陈少欣尹升明吴运杰杨松柏
Owner SHANGHAI INST OF PHARMA IND CO LTD
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