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A method for increasing the yield of nerolidol in Saccharomyces cerevisiae

A technology of tertiary nerolidol and Saccharomyces cerevisiae, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems such as the decline of enzyme gene transcription level, achieve the promotion of synthesis, increase of yield, increase of transcription activity and The effect of strain viability

Active Publication Date: 2021-04-06
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the present invention, we found that with the continuous increase of the number of gene expression cassettes driven by the GAL promoter integrated in the genome, the transcription level of the enzyme gene of the target product metabolic pathway in the secondary growth induction system will be greatly reduced

Method used

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  • A method for increasing the yield of nerolidol in Saccharomyces cerevisiae
  • A method for increasing the yield of nerolidol in Saccharomyces cerevisiae
  • A method for increasing the yield of nerolidol in Saccharomyces cerevisiae

Examples

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Effect test

Embodiment 1

[0027] Example 1 Construction of strains based on secondary growth induction system and determination of nerolidol production

[0028] 1. Strain construction

[0029] There are many examples of synthesizing terpenoids in the metabolic engineering of Saccharomyces cerevisiae. Based on these reports, we strengthened the metabolic flow of the MVA pathway and strengthened the conversion of ethanol to acetyl-CoA, while weakening the synthesis pathway of squalene. Refer to the literature ( Coupling gene regulatory patterns to bioprocess conditions to optimizesynthetic metabolic modules for improved sesquiterpene production in yeast, Biotechnol. Carotenoid-basedphenotypic screen of the yeast deletion collection reveals new genes withroles in isoprenoid production, Metab.Eng.15(2013)174-183.) Constructed a series of Saccharomyces cerevisiae capable of synthesizing nerolidol driven by a secondary growth induction system The bacterial strain (table 1) of engineering bacterium, carries ...

Embodiment 2

[0059] Determination of Hac1 transcript level in the bacterial strain constructed in embodiment 2

[0060] According to the instructions of the TransZol Up kit (Quanshijin, China), RNA was extracted from strains YS004, YS006, YS011, YS015, YS018, YS031 and YS037 after 72 h of culture, RNA was quantified by NanoDrop 2000c (Thermo Fisher, USA), and then 500ng of total RNA was used as a template, and cDNA was synthesized by reverse transcription using a qPCR RT kit (Toyobo, Japan). Quantitative PCR assay was performed with Lightcycler 480II Real-TimePCR System (Roche, Switzerland) using SYBR GreenReal time PCR Master Mix (Toyobo, Japan). Gene-specific primers are listed in Table 3. Set the PCR program as 95°C for 30s, then 95°C for 5s, 57°C for 5s and 72°C for 15s for a total of 40 cycles. use 2 -ΔΔCT Method, calculate the expression levels of Hac1, Gal1, Gal4 and AcNES1 mRNA relative to the internal reference ACT1, the results are shown in figure 2 shown.

[0061] In the u...

Embodiment 3

[0066] Example 3 Effect of overexpressing Hac1 on the synthesis of nerolidol

[0067] The primers and templates used in this example are listed in Table 4, and the constructed strains are listed in Table 5.

[0068] Construction of strain YS038: We constructed nerolidol synthesis by constitutively overexpressing Hac1 on the pRS316 plasmid (A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics (1989) 122, 19–27) Strain YS038. Use primers Hac1-F1 / Hac1-R1 and Hac1-F2 / Hac1-R2 in Table 4 to clone two DNA fragments of Hac1 from the genome of Saccharomyces cerevisiae CEN.PK2-1D, the first fragment (fragment 1) is SEQ ID 1-660 bases of NO.1, and the second fragment (fragment 2) is 661-991 bases of SEQ ID NO.1 (the last 274 bp is the terminator sequence of the HAC1 gene). The two DNA fragments of Hac1 were fused by overlap extension PCR to obtain the spliced ​​HAC1 fragment, i.e. sequence SEQID NO.1; using ...

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Abstract

The invention discloses a method for improving the production of nerolidol in Saccharomyces cerevisiae, which comprises: using a Gal promoter to drive the synthesis of mevalonate pathway metabolic enzymes and nerolidol in Saccharomyces cerevisiae with Gal80 gene knockout The expression of the enzyme forms a secondary growth induction system, and at the same time overexpresses the transcription factor hac1 gene to construct a yeast engineered bacterium that synthesizes nerolidol. The present invention uses the yeast secondary growth induction system for metabolic transformation. When the expression cassettes driven by the GAL promoter are integrated in the genome in large quantities, the transcriptional activity of the GAL promoter is greatly reduced, while the overexpression of the transcription factor Hac1 in the present invention can improve The transcriptional activity of the GAL promoter and the vigor of the strain can further promote the synthesis of target metabolites in the secondary growth induction system, and the yield of nerolidol increased by 48.4%, reaching 497mg / L.

Description

[0001] (1) Technical field [0002] The invention relates to a method for increasing the yield of nerolidol in Saccharomyces cerevisiae. [0003] (2) Background technology [0004] Saccharomyces cerevisiae has the advantages of simple genetic manipulation, high biological safety and stable fermentation, and is a widely used biological cell factory. The common C5 precursors of terpenoids are isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are synthesized in yeast via the mevalonate (MVA) pathway. By increasing the levels of acetyl-COA and MVA, researchers have successfully engineered Saccharomyces cerevisiae to be a high-yield microorganism for heterologous biosynthesis of terpenoids. Nerolidol is an aromatic sesquiterpene essential oil component widely used in cosmetics and washing. In addition, nerolidol also has antiviral, antitumor and antimalarial effects. The invention provides a new method for improving the production of yeast sesquiterpe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/04C12N15/81C12N1/19C12R1/865
CPCC07K14/395C12N9/0006C12N9/1025C12N9/1029C12N9/1085C12N9/1205C12N9/1229C12N9/88C12N15/81C12P7/04C12Y101/01034C12Y203/01009C12Y203/0301C12Y205/0101C12Y207/01036C12Y207/04002C12Y401/01033C12Y402/03048
Inventor 孙杰屈朕朕张丽丽竺少铭孔望欣王红卫夏美芳汪钊袁围郑建永
Owner ZHEJIANG UNIV OF TECH
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