Yeast genetically engineered strain for high yield of citronellol as well as construction method and fermentation method of yeast genetically engineered strain

The technology of a genetically engineered strain and a construction method, which is applied to a yeast genetically engineered strain with high citronellol yield and its construction field, can solve the problems of no CPP found, only the verification of enzyme function, low catalytic efficiency, etc., so as to reduce monoterpenes. Cytotoxic, low-cost effect

Active Publication Date: 2021-03-26
TIANJIN UNIV
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Problems solved by technology

It was first discovered in 1999 that Pelargonium graveolens can convert citronellyl pyrophosphate (CPP) into rose ether. The structure of rose ether is highly similar to citronellol, so they deduced that CPP can be used as a substrate to generate Citronellol, but CPP and CPP-related enzymes have not been found in plants
In 2019, Zhang Xianan's team from Capital Medical University discovered for the first time a gene that can catalyze citronellol from GPP (geranyl pyrophosphate) in vitro by analyzing the genome of Tripterygium wilfordii plant, but the catalytic efficiency is very low
In 2011 and 2013, two domestic and foreign teams respectively confirmed that the endogenous old yellow enzyme OYE2 of Saccharomyces cerevisiae can reduce geraniol to citronellol, but it is limited to the verification of the enzyme function

Method used

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  • Yeast genetically engineered strain for high yield of citronellol as well as construction method and fermentation method of yeast genetically engineered strain
  • Yeast genetically engineered strain for high yield of citronellol as well as construction method and fermentation method of yeast genetically engineered strain
  • Yeast genetically engineered strain for high yield of citronellol as well as construction method and fermentation method of yeast genetically engineered strain

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1 screening different reductases for synthesizing citronellol

[0049] 1. Acquisition of yeast chassis strains that strengthen the mevalonate pathway

[0050] Provided by Yuan Yingjin's research group, the strain number is SyBE_Sc02020001, the genotype of the strain is CEN.PK2-1C, gal80Δ::tHMG-P GAL1,10 -IDI1-His3.

[0051] 2. Construction of geraniol producing strains

[0052] The production of geraniol is catalyzed by vinca-derived geraniol synthase (CrGES) ( figure 1 ), in order to improve the catalytic efficiency of CrGES in Saccharomyces cerevisiae, its N-terminus was truncated by 43 amino acids, and the gene encoding CrGES was codon-optimized in Saccharomyces cerevisiae. t43CrGES and the gene ERG20 encoding the mutated farnesene synthase were synthesized by overlap extension PCR (OE-PCR). F96W,N127W Fusion together, the two genes are connected by a short flexible linker GSG to obtain the gene t43CrGES-ERG20 F96W,N127W (GE), BsaI enzyme cutting site...

Embodiment 2

[0065] Embodiment 2 The impact of transforming the chassis competition path on the output of citronellol

[0066] 1. The transformation method of competition path

[0067] Although overexpression of ERG20 F96W,N127W can make more precursors flow to citronellol, but wild-type ERG20 (FPP synthase gene) in S. cerevisiae chassis cells still competitively metabolizes the citronellol precursor GPP. In the present invention, ERG20 is mutated into ERG20 in situ by CRISPR-Cas9 technology F96W ( Figure 4). The gRNA used here is GGACTTGTCCATCATATCAT. It was ligated into the Cas9 plasmid digested with BsmBI by primer annealing. When constructing homologous fragments, the OE-PCR method was used to mutate the CGG upstream of the position corresponding to the gRNA on ERG20 to CAG to block the repeated cutting of Cas9. The mutation here is a synonymous mutation, and the 99-position alanine involved is not changes happened. Similarly, the introduction of F96W is also through the method...

Embodiment 4

[0082] Embodiment 4 Fermentation design and optimization of citronellol high-yielding bacterial strain

[0083] 1. Construction of integrated strains

[0084] The endogenous acetyltransferase ATF1 of Saccharomyces cerevisiae can metabolize the substrate geraniol and the product citronellol, so the expression cassette of IDI1-SH3 and SF1 were integrated into the ATF1 site, and the primers were designed to amplify P GAL7 -SF1-T GPD and T FBA1 -P GAL1 -IDI1-SH3-T TDH2 , A 50-60bp homologous sequence with ATF1 was added to the primer as an integrated homologous arm. The PCR product was recovered by DNA gel electrophoresis, and the two fragments were recovered and integrated into the ATF1 position on the SyBE_Sc02020227 genome through the CRISPR-Cas9 system, and the gRNA used was GACTTCGGAATAAACAAGTA. After primary screening by colony PCR, the integrated fragment was amplified with high-fidelity enzymes, and the correct clone was obtained after sequencing to verify that there ...

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Abstract

The invention relates to the technical field of saccharomyces cerevisiae strain construction and fermentation, in particular to a yeast genetically engineered strain for high yield of citronellol as well as a construction method and fermentation method of the yeast genetically engineered strain. The transformation of the yeast genetically engineered strain comprises the following steps: transferring one or more of CrIS, NmIS2, OeIS, OYE2 and OYE3 genes; mutating ERG20 of a yeast chassis strain into ERG20F96W; and introducing a protein scaffold consisting of SH3, PDZ and GBD while isomerase IDI1, reductase CrIS and fusion protein GE are expressed, so that a protein complex is assembled. Compared with the prior art, the recombinant saccharomyces cerevisiae strain is more environmentally-friendly and low in cost, and a green and efficient method is provided for citronellol production.

Description

technical field [0001] The invention relates to the technical field of construction and fermentation of Saccharomyces yeast strains, in particular to a yeast genetically engineered strain with high yield of citronellol and its construction method and fermentation method. Background technique [0002] Citronellol (Citronellol, 3,7-dimethyl-6-octen-1-ol, C 10 h 20 O, with a molecular weight of 156.27), is a chiral linear monoterpene compound, which is divided into three types: L-citronellol (rose alcohol), D-citronellol and racemic citronellol. Citronellol exists in a variety of plant volatile oils, such as citronella oil, rose oil, rue oil, etc. Because of its sweet and elegant smell, it occupies a broad market in the field of flavors and fragrances, with an annual demand of more than 1,000 tons. It can be used in cosmetics, skin care products, bath products, and household cleaning products. Citronellol can also be used in the agricultural field and has a good insecticidal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/04C12R1/865
CPCC12N15/52C12N9/0036C12N9/001C12N9/90C07K14/47C12N15/81C12P7/04C12Y106/99001C12Y103/01092C12Y103/01C12Y503/03002C12N2800/22Y02E50/10
Inventor 元英进姜国珍肖文海姚明东王颖
Owner TIANJIN UNIV
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