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Saffron crocus sourced CCD2 mutant, coding sequence and application thereof, as well as recombinant yeast strain for producing crocetin

A coding sequence and yeast strain technology, which is applied in the field of genetic engineering, can solve the problems of catalyzing beta-carotene and lycopene, reducing the total yield, and adapting the pathway gene to the chassis, etc., so as to solve the problems of generally low total yield, Improve efficiency and shorten the effect of synthesis paths

Active Publication Date: 2019-05-28
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] CCD2 derived from saffron is the only CCD family member found so far that can catalyze the synthesis of crocetin from zeaxanthin in Saccharomyces cerevisiae. The identified substrate spectrum does not include β-carotene and lycopene; at present, although the synthesis of crocetin in S. The heterologous synthesis of long-path natural products of acid faces the problem of adaptation between genes in the pathway and between genes in the pathway and the chassis
At the same time, with the extension of the route, the total yield continues to decrease, and the yield is still very low

Method used

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  • Saffron crocus sourced CCD2 mutant, coding sequence and application thereof, as well as recombinant yeast strain for producing crocetin
  • Saffron crocus sourced CCD2 mutant, coding sequence and application thereof, as well as recombinant yeast strain for producing crocetin
  • Saffron crocus sourced CCD2 mutant, coding sequence and application thereof, as well as recombinant yeast strain for producing crocetin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: Construct recombinant Saccharomyces cerevisiae strain of the present invention

[0070] 1. Chassis strains

[0071] Saccharomyces cerevisiae strains or common Saccharomyces cerevisiae strains capable of high production of β-carotene and / or zeaxanthin can be selected (common strains need to add β-carotene and / or zeaxanthin as substrates during fermentation);

[0072] 2. Acquisition of exogenous functional gene elements and modular integration tool plasmids

[0073] CCD2 gene element: obtained by amplifying the pRS416-C-01 plasmid as a template (there is an expression cassette HIS5t-GAL10p-CCD2-TEF2t on it);

[0074] SynALD gene element: obtained by amplifying the pRS425K-A-04 plasmid as a template (there is an expression cassette TEF2t-GAL7p-SynALD-PGI1t on it);

[0075] The basic vector is the pRS416-A-01 plasmid, which has the HIS5t-PGI1t gene element;

[0076] The above-mentioned genetic elements, plasmids or expression cassettes can also be obtained ...

Embodiment 2

[0084] Embodiment 2: the crocetin shake flask fermentation test (beta-carotene substrate) of the recombinant Saccharomyces cerevisiae of CCD2 single point mutation

[0085] 1. Test strain

[0086] The chassis strains of each group are SyBE_Sc14CY03 high-yielding β-carotene strains in patent 201510435606.2.

[0087] Control strain 1: constructed with reference to the construction method in Example 1, the difference is that the wild-type CCD1 gene is used to replace the CCD2 gene, that is, the difference is that the expression cassette one is HIS5t-GAL10p-CCD1-TEF2t, and the strain number is SyBE_Sc02070246;

[0088] Control strain 2: constructed with reference to the construction method in Example 1, the difference is that the CCD2 gene is a wild-type unmutated gene, that is, the difference is that the expression cassette one is HIS5t-GAL10p-CCD2-TEF2t;

[0089] Recombinant strain 1 of the present invention: the site mutation is selected as S323F, that is, the expression casse...

Embodiment 3

[0099] Embodiment 3: the crocetin shake flask fermentation test (beta-carotene substrate) of the recombinant Saccharomyces cerevisiae of CCD2 multi-point mutation

[0100] Construct recombinant bacterial strain according to the construction method of embodiment 1, chassis bacterial strain is the same as embodiment 2, select following CCD2 multi-point mutation, each group expression cassette one is as follows:

[0101] HIS5t-GAL10p-CCD2_mut24(R192F&S323F)-TEF2t;

[0102] HIS5t-GAL10p-CCD2_mut25(R192F&S323A)-TEF2t;

[0103] HIS5t-GAL10p-CCD2_mut26(T290V&S323F)-TEF2t;

[0104] HIS5t-GAL10p-CCD2_mut27(T290V&S323A)-TEF2t;

[0105] HIS5t-GAL10p-CCD2_mut28(R192F&T290V&S323F)-TEF2t;

[0106] HIS5t-GAL10p-CCD2_mut29(R192F&T290V&S323A)-TEF2t.

[0107] The strain numbers are SyBE_Sc02070264-SyBE_Sc02070269;

[0108] see results Figure 7 , Combining the S323A mutation with other mutations can significantly increase the production of crocetin in recombinant S.

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Abstract

The invention relates to the technical field of genetic engineering, and discloses a saffron crocus sourced CCD2 mutant, a coding sequence and application thereof, as well as a recombinant yeast strain for producing crocetin. The CCD2 mutant is provided with one or two or above of V120F, Y190K / A, R192V / F, E211A, E212A, T290V, K320A and S323F / A / T locus mutations. A rate-limiting enzyme CCD2 in thesynthesis route of the crocetin is transformed, the crocetin is directly synthesized from beta-carotene by widening the substrate spectrum of the CCD2, and the existing synthesis route of the crocetinis simplified and reconstructed, so that the problem that the total yield of multiple reactions is generally low is solved; and meanwhile, the efficiency of the CCD2 catalyzing an original substratezeaxanthin to synthesize the crocetin is improved, and the yeast strain of the high-yield crocetin is further obtained.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a CCD2 mutant derived from saffron, its coding sequence and application, and a recombinant yeast strain producing crocetin. Background technique [0002] Crocetin, also known as crocetin or crocetin, is one of the active ingredients of saffron extract, and its molecular formula is C 20 h 24 o 4 . The main source of saffron acid is the stigma of saffron, but the resources of saffron in nature are very limited, and its stigma production is extremely low. Each saffron can only produce three pistil stigmas in its lifetime. An average of 250,000 saffron flowers, it takes 40 hours of manual extraction to obtain enough stigmas to produce 1kg of saffron powder (saffron), and the market price of saffron acid with a content of 97% is as high as 136,000 yuan / g, so saffron enjoys the "plant gold" reputation. [0003] It is recorded in Chinese classical medical books that saf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P11/00C12R1/865
Inventor 元英进梁楠姚明东王颖肖文海
Owner TIANJIN UNIV
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