Mutant strains for realizing universal enzyme catalytic function diversity and construction method thereof

A construction method and mutant technology are applied in the field of mutant strains that realize the diversity of universal enzyme catalytic functions and the field of construction thereof, which can solve the problems of interspersed three-step dehydrogenation product streptavidin and the like, and achieve controllability and enrichment. The effect of functional information

Active Publication Date: 2019-06-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, CrtI derived from Blakeslea trispora showed better substrate conversion efficiency, but still contained a small amount of three-step dehydrogenation product streptosporine

Method used

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  • Mutant strains for realizing universal enzyme catalytic function diversity and construction method thereof
  • Mutant strains for realizing universal enzyme catalytic function diversity and construction method thereof
  • Mutant strains for realizing universal enzyme catalytic function diversity and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Acquisition of Saccharomyces cerevisiae producing high phytoene

[0041] 1. Construction of modular integration plasmids

[0042] The Saccharomyces cerevisiae strain with high GGPP production, the strain number is SyBE_Sc14C10 (Chen Yan; Design, construction and fermentation process optimization of Saccharomyces cerevisiae with high lycopene production [D]; Tianjin University; 2017).

[0043] In order to realize the production of phytoene in Saccharomyces cerevisiae, elements such as yeast promoter Gal1-10, gene CrtB, terminator Pgk1t, upstream integrated homology arm, and downstream integrated homology arm were obtained by PCR amplification. CrtB was optimized according to the codons of Saccharomyces cerevisiae and was synthesized by Suzhou Jinweizhi Co., Ltd. after properly avoiding common restriction enzyme sites. The above elements were modularly assembled by overlapping extension PCR (OE-PCR) to obtain L-arm-P GAL1 -CrtB-Tpgk1-R-arm was named Module1 (S...

Embodiment 2

[0046] Embodiment 2, the construction of wild-type BtCrtI in Saccharomyces cerevisiae

[0047] 1. Construction of a Saccharomyces cerevisiae strain that catalyzes phytoene to produce lycopene with CrtI ( image 3 )

[0048] The GAL7 promoter, the CYC1t terminator, and the gene BtCrtI synthesized by Suzhou Jinweizhi Company according to the codons of Saccharomyces cerevisiae, which were optimized according to the codon of S. The linear fragment of the NotI restriction site GAL7 promoter-BtCrtI-CYC1t terminator, that is, PGAL7-BtCrtI-TCYC1 (SEQ ID NO.2); Afterwards, the linear fragment and the PRS416 plasmid were treated with XbaI and NotI endonucleases , connected with T4 ligase, transformed into E. coli competent DH5α, screened by colony PCR, extracted plasmids for single and double enzyme digestion verification and sequencing verification to ensure that the target fragments are connected correctly and the base sequence is not mutated, so that the construction is correct rec...

Embodiment 3

[0057] Embodiment 3, the construction of wild-type BtCrtI in Saccharomyces cerevisiae

[0058] 1. Construction of BtCrtI mutation library by error-prone PCR

[0059] experiment material:

[0060] Strain SyBE_Sc04020001 with plasmid pRS416-BtCrtI

[0061] 10× error-prone PCR buffer: 70mm magnesium chloride hexahydrate, 500mm potassium chloride, 100mm tris, 0.1w / v gelatin

[0062] 10× error-prone PCR dNTPs: 10mm dCTPs and dTTPs, 2mm dGTPs and dATPs

[0063] 10mm manganese chloride tetrahydrate

[0064] The GAL7 promoter and CYC1t terminator were amplified by PCR, and the BtCrtI gene was amplified by random mutation of error-prone PCR using the plasmid containing BtCrtI as a template. The error-prone PCR system (100 μl) was configured as follows: add 10× PCR buffer 10 μl, 10× error-prone PCR dNTPs 10 μl, upper and lower primers 4 μl, template 2 μl, manganese chloride solution 5 μl, fasttaq enzyme 1 μl, and supplemented water 64 μl.

[0065] It should be noted that there is a...

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Abstract

The invention belongs to the field of microorganisms, and discloses a construction method of mutant strains for realizing universal enzyme catalytic functional diversity, which comprises the followingsteps of: constructing a mutant library of a universal enzyme; and screening the mutant strains with catalytic functional diversity through shake flask fermentation. By means of the method, a recombinant saccharomyces cerevisiae mutant strain which generates zeta-carotene by BtCrtI catalytic two-step dehydrogenation reaction and a recombinant saccharomyces cerevisiae mutant strain with the ratioof a lycopene produced by catalytic four-step dehydrogenation and a neurosporene produced by three-step dehydrogenation reaction is 5:1..By means of the method, the recombinant saccharomyces cerevisiae strain is utilized to obtain mutant strains of BtCrtI with different catalytic functions through a random mutation property, key amino acid sites are determined, the controllability of dehydrogenation steps is realized, the functional information of CrtI is enriched, and a foundation is laid for the biosynthesis of important natural products.

Description

technical field [0001] The invention belongs to the field of microorganisms, and specifically relates to a mutant strain and a construction method thereof for realizing the diversity of general enzyme catalytic functions, in particular to a recombinant Saccharomyces cerevisiae mutant strain and a recombinant Saccharomyces cerevisiae mutant strain that produces zeta-carotene through a two-step dehydrogenation reaction catalyzed by BtCrtI. A recombinant Saccharomyces cerevisiae mutant strain with a ratio of 5:1 of lycopene produced by catalyzing four-step dehydrogenation and streptosporine produced by three-step dehydrogenation and its construction method. Background technique [0002] In the process of biological metabolism, there exists a class of universal enzymes that can catalyze multi-step continuous reactions. According to statistics, about 20% of biological metabolic enzymes can be classified as general-purpose enzymes, which play an important role in the metabolic pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12N15/81C12N15/66C12R1/865
Inventor 元英进陈琛姚明东王颖肖文海
Owner TIANJIN UNIV
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