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Method for improving linalool synthesizing capacity of saccharomyces cerevisiae

A technology for Saccharomyces cerevisiae and linalool, applied in the field of metabolic engineering, can solve the problems of unstable expression of microorganisms, low yield of linalool, etc., and achieve the effects of increasing substrate supply, improving fermentation yield, and realizing ability

Inactive Publication Date: 2018-04-20
INT CENT FOR BAMBOO & RATTAN
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, scholars at home and abroad have made in-depth research on the production of linalool by fermentation of Saccharomyces cerevisiae, but there are still problems such as unstable microbial expression and low yield of linalool.

Method used

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  • Method for improving linalool synthesizing capacity of saccharomyces cerevisiae
  • Method for improving linalool synthesizing capacity of saccharomyces cerevisiae
  • Method for improving linalool synthesizing capacity of saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The culture medium used in the present invention comprises as follows:

[0020] (1) YPD medium: 1 g of yeast extract, 2 g of peptone, 2 g of glucose, 100 mL of deionized water, mixed well, and sterilized at 115° C. in a sterilizing pot.

[0021] (2) Amino acid solution used in the fermentation medium: Weigh the following amino acids, mix them, and dissolve them in sterilized water to make the final concentration: L-Adenine hemisulfate salt (L-Adenine hemisulfate salt) 200mg / L, L-arginine hydrochloride (L-Arginine HCl) 200mg / L, L-histidine hydrochloride monohydrate (L-Histidine HCI monohydrate) 200mg / L, L-isoleucine (L-Isoleucine ) 300mg / L, L-leucine (L-Leucine) 1000mg / L, L-lysine hydrochloride (L-Lysine HCI) 300mg / L, L-methionine (L-Methionine) 200mg / L, L -Phenylalanine (L-Phenylalanine) 500mg / L, L-Threonine (L-Threonine) 2000mg / L, L-Tryptophan (L-Tryptophan) 200mg / L, L-Tyrosine (L- Tyrosine) 300mg / L, L-valine (L-Valine) 1500mg / L. The amino acid mother liquor was ste...

Embodiment 2 3

[0040] The construction of embodiment 2 three kinds of expression vectors and comparative effect

[0041] In order to improve the fermentation yield of linalool, three different expression vectors were screened and compared (1) pESC-URA-CoLIS (linalool gene); (2) pESC-URA-CoLIS::ERG20 (fusion gene); (3) pESC-URA-CoLIS / ERG20 (co-expressed gene).

[0042] 1. Preparation of Linear Vectors

[0043] ●The expression vector pESC-URA plasmid was digested with speI / EcoRI to prepare a linear vector. The size of the linearized vector was about 6.5kb, and the gel was cut and recovered.

[0044] ●The expression vector pESC-URA-LIS plasmid was digested with BamHI / SaI1 to prepare a linear vector. The size of the linearized vector was about 8.2kb, and the gel was cut and recovered.

[0045] 2. Target fragment amplification and vector construction

[0046] ●Construction of pESC-URA-LIS vector: Primer LIS-F / LIS-R amplifies the LIS fragment, the fragment length is 1755bp, and it is constructe...

Embodiment 3

[0060] The detection of embodiment 3 linalool

[0061] The recombinant strain was cultured in a liquid medium galactose medium (SG-URA) in a shaker flask (take 0.5 of the bacterial liquid (seed liquid) of the above-mentioned recombinant bacteria in 50 mL of SG-URA medium) and cultivate for 3 days. The production of linalool was detected by GC-MS by headspace sampling. Column thermostat temperature program mode: initial temperature 50°C, keep for 1min, increase temperature at 5°C / min to 115°C and hold for 1min; inlet temperature 240°C; mass spectrometry conditions: electron bombardment (EI) ion source; electron energy 70eV; transmission line The temperature is 280°C; the ion source temperature is 230°C; the quadrupole temperature is 150°C. The results showed that the vector (pESC-URA-LIS / ERG20) constructed by gene co-expression, after transforming S. The content of linalool reaches 3.5mg / L in the liquid.

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Abstract

The invention discloses a method for improving a linalool synthesizing capacity of saccharomyces cerevisiae, and belongs to the technical field of metabolic engineering. The method comprises the following steps: firstly, conducting codon optimization on a CoLIS gene sequence SEQ ID NO:1 from cinnamomum pedumculatum nees and an ERG20 gene sequence SEQ ID NO:2 from the saccharomyces cerevisiae; on the basis of enzyme digestion and connection, cloning the SEQ ID NO:1 and the SEQ ID NO:2 to a plasmid pESC-URA; and conducting regulating and controlling by virtue of double promoters, namely Gal10 and Gal1, on a pESC-URA vector, so that activities of CoLIS and ERG 20 enzyme protein are reserved; through high-expression of the ERG20 protein, a substrate, namely geranyl pyrophosphate (GPP), which is required by the synthesis of linalool, can be effectively supplied by virtue of recombinant bacteria; therefore, an enzymatic reaction rate for synthesizing linalool can be obviously improved and fermentation conditions can be further optimized, so that the effective synthesis of the linalool is achieved.

Description

technical field [0001] The invention relates to a method for improving the ability of Saccharomyces cerevisiae to synthesize linalool, which belongs to the technical field of metabolic engineering. Background technique [0002] Linalool (Linalool) belongs to terpene alcohol compounds, and its chemical name is 3,7-dimethyl-1,6-octadien-3-ol. It has been widely used in daily fragrances, medicine and other fields. The global annual demand for linalool reaches 50,000 tons, more than 90% of which come from chemical synthesis, and chemical synthesis brings serious environmental pollution. Therefore, the preparation of linalool by green and pollution-free biosynthetic methods has become an important research direction. [0003] As a recognized safe model microorganism, Saccharomyces cerevisiae has a relatively clear mechanism of gene expression regulation and is easy to operate, so it is used in biosynthesis research or production. Yu Hongwei's research group at Zhejiang Universi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/31C12N15/60C12R1/865
CPCC07K14/395C12N9/88C12N15/81C12N2800/22C12Y402/03
Inventor 王进曹先爽汤锋王煜炜荀航
Owner INT CENT FOR BAMBOO & RATTAN
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