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Saccharomyces cerevisiae for producing vitamin A and construction method thereof

A technology of Saccharomyces cerevisiae and vitamins, applied in the field of fermentation engineering, can solve problems such as the failure to achieve a breakthrough in production, low expression of rate-limiting enzymes, and complex metabolic pathways

Pending Publication Date: 2022-05-13
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For this reason, the technical problem to be solved by the present invention is to overcome the insufficient supply of endogenous isoprenoid precursors and the low expression of rate-limiting enzymes related to exogenous carotenoid synthesis pathways when Saccharomyces cerevisiae synthesizes vitamin A in the prior art And the complex metabolic pathways lead to the failure to achieve a breakthrough in yield and other defects

Method used

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  • Saccharomyces cerevisiae for producing vitamin A and construction method thereof
  • Saccharomyces cerevisiae for producing vitamin A and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1. Construction of recombinant bacteria A1

[0054] Based on the expression preference of Saccharomyces cerevisiae, geranylgeranyl diphosphate synthase (GGPP synthase, crtE), phytoene synthase (crtB), phytoene desaturase ( crtI), lycopene cyclase (crtYB) and β-carotene 15,15′-monooxygenase (BCMO) gene fragments, wherein Crt E derived from Taxus mandia (Taxus x media), Crt B from Pantoea agglomerans, Crt I from Blakeslea trispora, crtYB from Xanthophyllomyces Dendrorhous and marine bacteria 66A03 (uncultured marine BCMO of bacterium 66A03) is highly expressed in Saccharomyces cerevisiae.

[0055] The specific construction process is as follows:

[0056] (1) Synthetic gene fragment T ADH1 -crtE-P GAL10 -crtI-T CYC1 , using the Saccharomyces cerevisiae BY4741 genome as a template, using primers 208a-U-F, 208a-U-R to amplify gene 208a-U, using primers 208a-D-F, 208a-D-R to amplify gene 208a-D, using pMHyLp-Trp plasmid as a template , using primers loxT-1F, ...

Embodiment 2

[0070] Embodiment 2. Construction of recombinant bacteria A2

[0071] The specific construction process is as follows:

[0072] (1) Artificially synthesized gene fragment P GAL7 -crtB-T AOX1 . Using the Saccharomyces cerevisiae BY4741 genome as a template, using primers 1021b-U-F and 1021b-U-R to amplify gene 1021b-U, using primers 1021b-D-F and 1021b-D-R to amplify gene 1021b-D, using pMHyLp-His plasmid as a template, The loxH-1 gene fragment was amplified by primers loxH-1F and loxH-1R.

[0073] (2) the three gene fragments obtained in step (1) P GAL7 -crtB-T AOX1 , 1021b-U and 1021b-D for overlap extension PCR, the PCR conditions are 98°C, 5min pre-denaturation, then 98°C, denaturation 10s, 55°C, annealing 5s, 7°C, extension 2min, a total of 30 cycles, 1% After the agarose gel electrophoresis was verified correctly, the fragment was recovered by cutting the gel to obtain the fusion gene fragment 1021b-U-P GAL7 -crtB-T AOX1 -1021b-D gene fragment.

[0074] (3) Prepa...

Embodiment 3

[0085] Embodiment 3. Construction of recombinant bacteria A3

[0086] The specific construction process is as follows:

[0087] (1) Artificially synthesized gene fragment P GAL7 -crtYB-T AOX1 , using the Saccharomyces cerevisiae BY4741 genome as a template, using primers 1622b-U-F, 1622b-U-R to amplify gene 1622b-U, using primers 1622b-D-F, 1622b-D-R to amplify gene 1622b-D, using pMHyLp-Leu plasmid as a template , using primers loxL-1F and loxL-1R to amplify the loxL-1 gene fragment.

[0088] (2) the three gene fragments obtained in step (1) P GAL7 -crtYB-T AOX1 , 1622b-U and 1622b-D for overlap extension PCR, the PCR conditions are 98°C, 5min pre-denaturation, then 98°C, denaturation 10s, 55°C, annealing 5s, 7°C, extension 2min, a total of 30 cycles, 1% After the agarose gel electrophoresis was verified correctly, the fragment was recovered by cutting the gel to obtain the fusion gene fragment 1622b-U-P GAL7 -crtYB-T AOX1 -1622b-D gene fragment.

[0089] (3) the fusi...

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Abstract

The invention relates to saccharomyces cerevisiae for producing vitamin A and a construction method of the saccharomyces cerevisiae, and belongs to the technical field of fermentation engineering. The saccharomyces cerevisiae genetically engineered bacterium is obtained by taking saccharomyces cerevisiae as an original strain, constructing a vitamin A synthesis pathway in yeast cells, overexpressing endogenous vitamin A of the saccharomyces cerevisiae to synthesize a mevalonic acid pathway of a precursor isoprene pyrophosphate, and strengthening key gene expression of an exogenous carotenoid synthesis pathway of the saccharomyces cerevisiae. When the saccharomyces cerevisiae genetically engineered bacterium is used for fermentation production of the vitamin A, the shake flask yield can reach 193.9 mg / L, and the yield of the vitamin A is obviously improved.

Description

technical field [0001] The invention relates to the technical field of fermentation engineering, in particular to a vitamin A-producing Saccharomyces cerevisiae and a construction method thereof. Background technique [0002] Vitamin A (VitaminA, VA), known as "beauty vitamin" and "anti-dry eye vitamin", is an essential micronutrient for the human body, cannot be synthesized in the body, and needs to be obtained through exogenous means (mainly diet). The main biologically active forms of vitamin A are retinol, retinal and retinoic acid, among which retinol mainly exists in animal liver and is the main naturally occurring form of vitamin A. [0003] Vitamin A can be extracted from animal tissues, but the resources are scattered, the steps are complicated, and the cost is high. At present, commercial vitamin A is a chemically synthesized product, and the key intermediate citral production technology lacks control. Most of the upstream intermediate supply of the vitamin indust...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P23/00A23L31/10C12R1/865
CPCC12N9/1085C12N9/90C12N9/001C12N9/0083C12Y114/99036C12Y205/01029C12Y101/01034C12N9/0006C12Y503/03002C12Y205/0101C07K14/395C12Y205/01021C12P23/00A23L31/10A23V2002/00A23V2250/762
Inventor 刘龙石勇陈坚石训吕雪芹张攀攀堵国成李江华刘延峰王静王青霞王璇刘家恒
Owner JIANGNAN UNIV
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