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Yeast for producing D-limonene and application thereof

A limonene, yeast technology, applied in microorganism-based methods, fermentation, enzymes, etc., to achieve the effect of promoting redox balance, improving pathway efficiency, and improving growth rate

Active Publication Date: 2022-06-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]In summary, the traditional separation and extraction technology of limonene has problems such as low yield and high energy consumption, and is limited by raw materials and regions
Regarding the technologies and methods of synthetic biology, the current production of limonene in existing domestic and foreign technologies cannot meet the requirements of industrial applications
Monoterpenes tend to adhere to or permeate the cell membrane, which eventually leads to impairment of yeast membrane function, so there is also the problem of inhibition of yeast growth by limonene

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of D-limonene synthesis pathway

[0032] (a) Artificially synthesized gene fragment P GAL1 -tLimS-T CYC1 -P GAL10 -ERG20 WW -T ADH1 , wherein the tLimS gene is formed by removing the first 56 amino acid sequences of the gene LIS, ERG20 WW The gene is obtained by mutating the 96 and 127 amino acids of the ERG20 gene, and the above fragments are amplified by using primers 911b-F and 911b-R; the gene fragment 911b-U (primer 911b -U-F, 911b-U-R), gene fragment 911b-D (primers 911b-D-F, 911b-D-R);

[0033] (b) Using the nucleases SwaI and BelI * Digest the pML104 plasmid to obtain a linearized plasmid fragment;

[0034] (c) Gradient annealing with primers 911b-oligo2-F and 911b-oligo1-R. The specific method is to add the above two primers to the system at a ratio of 1:1, set the temperature of the PCR instrument from 98°C to 12°C, and 0.1°C rate annealing;

[0035] (d) Ligate the linearized plasmid fragment obtained in (b) with the annealed pr...

Embodiment 2

[0054] Example 2 Optimizing the endogenous MVA pathway

[0055] (a) Using the S288C genome of Saccharomyces cerevisiae as a template, artificially synthesized gene fragment P GAL1 -tHMG1-T ADH1 -P GAL10 -IDI-T CYC1 , and then amplified with primers GAL80-F and GAL80-R. Using the Saccharomyces cerevisiae S288C genome as a template, primers GAL80-U-F and GAL80-U-R were used to amplify the upstream 1000bp gene fragment of GAL80, and primers GAL80-D-F and GAL80-D-R were used to amplify the downstream 1000bp gene fragment of the gene GAL80.

[0056] (b) Refer to the method similar to steps (b) and (c) in Example 1, use the gradient annealing of primers GAL80-oligo2-F and GAL80-oligo1-R, and refer to the method similar to step (d) in Example 1 to construct The plasmid pML104-GAL80CRISPR-Cas9 was obtained.

[0057] (c) Prepare Saccharomyces cerevisiae MKL1 competent, and the amplified GAL80 upstream and downstream 1000bp gene fragment, gene fragment P GAL1 -tHMG1-T ADH1 -P GA...

Embodiment 3

[0118] Example 3 Optimization of D-limonene synthesis pathway key gene copy number

[0119] (a) Using the S288C genome of Saccharomyces cerevisiae as a template, artificially synthesized gene fragment P GAL1 -ERG20 WW -T ADH1 -P GAL10 -tLimS-T CYC1, then use primers SAP155b-F, SAP155b-R to amplify it, use Saccharomyces cerevisiae S288C genome as a template, use primers SAP155b-U-F, SAP155b-U-R to amplify the upstream 500bp gene fragment of SAP155b, use primers SAP155b-D-F, SAP155b- D-R amplification obtained a 500bp gene fragment downstream of the integration site SAP155b.

[0120] (b) Refer to the method similar to step (b) (c) of Example 1, use primers SAP155b-oligo2-F and SAP155b-oligo1-R gradient annealing, refer to the method similar to step (d) of Example 1, construct and obtain plasmid pML104 - Plasmid of SAP155bCRISPR-Cas9.

[0121] (c) Prepare Saccharomyces cerevisiae MKL5 competent, and the amplified SAP155b upstream and downstream 500bp gene fragment, pML104-S...

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PUM

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Abstract

The invention belongs to the field of metabolic engineering, and particularly relates to saccharomyces cerevisiae for producing D-limonene and application of the saccharomyces cerevisiae. Saccharomyces cerevisiae is used as an original strain, limonene synthase genes which do not exist in the same organism in the nature are organically integrated into the saccharomyces cerevisiae, expression of exogenous genes is optimized, MVA pathway gene key genes are over-expressed, supply of acetyl coenzyme A and NADPH precursors is increased, efficient synthesis of limonene in the saccharomyces cerevisiae is achieved, and the limonene synthase genes are synthesized into the saccharomyces cerevisiae. The finally obtained bacterial strain has a better growth rate compared with the original bacterial strain. The limonene yield can reach 657 mg / L and is increased by 2.021 times compared with the original yield through shake flask fermentation, efficient synthesis of limonene in saccharomyces cerevisiae is achieved, and the method has high application and popularization value.

Description

technical field [0001] The invention belongs to the field of metabolic engineering, in particular to a yeast producing D-limonene and its application. Background technique [0002] Terpenoids, also known as isoprenoids, are composed of compounds composed of isoprene units and their derivatives, and are secondary metabolites of plants. Limonene is a monocyclic monoterpenoid with the chemical formula C 10 h 16 , is a natural active compound derived from plants, volatile, relatively stable, and has a wide range of applications. In the food industry, limonene has antiseptic, antibacterial, anti-oxidation and other functions; in the pharmaceutical industry, limonene can be used to prepare drugs with anti-tumor, expectorant and asthma, and choleretic and stone-dissolving functions; In the industry, limonene is used as essential oil, mild detergent, etc.; in agriculture, limonene can be used to prepare natural source pesticides, which has the advantage of being easy to degrade i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P5/00C12R1/865C12R1/84C12R1/645
CPCC12P5/002C12N9/88C12N9/16C12N9/0006C12N9/1205C12Y402/03016C12Y402/0302C12Y301/07006C12Y401/01033C12Y101/01088C12Y301/02011C12Y301/02005C12Y207/01036Y02E50/10
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰孔晓
Owner JIANGNAN UNIV
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