Recombinant yeast for increasing yield of L-menthol

A technology of improving yield and yeast, which is applied in the field of metabolic engineering, can solve the problems of long growth cycle of plant raw materials, slow market feedback, unstable yield, etc.

Active Publication Date: 2021-01-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the industrial production method of L-menthol is relatively single, mainly natural extraction and chemical synthesis. The natural extraction method requires a large amount of mint plant raw materials, and the chemical synthesis of L-menthol also requires compounds such as thymol from plant sources as substrates. Perform reaction splitting
However, the growth cycle of plant raw materials is long, the market feedback is slow, and it is easily affected by natural environmental factors such as climate and region, resulting in unstable output
In addition, there are methods for preparing L-menthol by whole cells or enzymatically catalyzing the preparation of L-menthol with long-leaved menthone and the like as substrates, which still cannot solve the limiting factors of plant raw materials
[0003] The applicant constructed a strain of recombinant Saccharomyces cerevisiae engineering strain WMT4 in the early stage, realized the fermentation method to produce L-menthol for the first time, and used cheap fermentation medium to cultivate for about 4 days, and the fermentation yield of L-menthol reached 5.03mg / L, but Still there is the shortcoming that L-menthol fermentation yield is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Optimizing the endogenous MVA pathway

[0020] (a) Using the genome of Saccharomyces cerevisiae S288C as a template, use primers tHMG1-F and tHMG1-R to amplify gene tHMG1, tHMG1 is formed by removing the first 530 amino acid sequences of gene HMG1, and use primers GPD-F and GPD-R to amplify The promoter gene GPD (SEQ ID NO.3) was obtained, and the terminator gene ADH1-1 (SEQ ID NO.4) was obtained by amplification with primers ADH1-F1 and ADH1-R1; the amplification with primers IDI-F and IDI-R To obtain gene IDI, the promoter gene TEF1 (SEQ ID NO.5) was obtained by using primer TEF1-F and TEF1-R amplification, and the terminator gene CYC1-1 (SEQ ID NO.5) was obtained by amplification of primer CYC1-F1 and CYC1-R1. .6), use primers 208a-D-F, 208a-D-R to amplify to obtain gene fragment 208a-D, and use primers 208a-U-F, 208a-U-R to amplify to obtain gene fragment 208a-F.

[0021] (b) divide the six fragments tHMG1, GPD, ADH1-1 and IDI, TEF1, CYC1-1 gene fragment...

Embodiment 2

[0046] Example 2: Reconstruction of L-menthol metabolic synthesis pathway

[0047] (a) Artificially synthesized gene fragment P GAL1 -tLIS -T ADH1 -P GAL10 -L3H-T CYC1 -P GAL7 -CPR-T TDH3 ,P GAL1 -IPDH-T ADH1 -P GAL10 -IPR-T CYC1 -P GAL7 -KSI-T TDH3 ,P GAL1 -PGR-T ADH1 -P GAL10 -MMR-T CYC1 , wherein the tLIS gene is formed by removing the first 56 amino acid sequences of the gene LIS. Using the S. cerevisiae S288C genome as a template, gene fragment 911b-U, gene fragment 911b-D, gene fragment 308a-U, gene fragment 308a-D, gene fragment 1622b-U and gene fragment 1622b-D were respectively amplified.

[0048] (b) Using the plasmid pML104 as a template, fragments 308a, 911b and 1622b were respectively amplified, and the plasmids pML104-308a, pML104-911b and pML104-1622b were constructed by using a method similar to the step in Example 1(c).

[0049] (c) prepare the competent state of bacterial strain WMT1, and the synthetic gene fragment P GAL1 -tLIS -T ADH1 ,P ...

Embodiment 3

[0074] Embodiment 3: Reconstructing Saccharomyces cerevisiae engineered strain WMT5

[0075] (a) Artificially synthesized gene fragment P GAL1 -ERG20WW-T ADH1 , using Saccharomyces cerevisiae S288C genome as a template, using primers 416d-U-F and 416d-U-R to amplify gene 416d-U, and using primers 416d-D-F and 416d-D-R to amplify gene 416d-D.

[0076] (b) the three fragments P obtained in step (a) GAL1 -ERG20WW-T ADH1 , 416d-U, and 416d-D gene fragments for overlap extension PCR, PCR conditions: 98°C, 5min pre-denaturation, then 98°C, denaturation 10s, 55°C, annealing 5s, 72°C, extension 2min, a total of 30 cycles, Cut the gel to recover the fragment of the correct size, and obtain the fusion gene fragment 416d-U-P GAL1 -ERG20WW-T ADH1 -416d-D gene fragment.

[0077] (c) Use the pML104 plasmid as a template, use primers 416d-F, 416d-R to amplify to obtain fragment 416d, after heat shock transfer into Escherichia coli JM109 competent, use primer 416d-YZ-F and L4440 colony PC...

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Abstract

The invention discloses recombinant yeast for increasing the yield of L-menthol, and belongs to the technical field of metabolic engineering. The yeast is used as a starting strain, an inducible promoter is adopted to express ERG20WW or / and NPPS genes, and an MVA metabolic synthesis path is reconstructed, so that compounds IPP and DMAPP tend to synthesize compounds GPP and NPP more, and metabolicsynthesis of the L-menthol is further facilitated. The inducible promoter is adopted for over-expression of an L3H gene, the expression quantity of an L3H enzyme is increased, the conversion of limonene into trans-isopiperitol is promoted, or tLIS and L3H genes are expressed through protease fusion, and the factor of the too low enzyme activity of the L3H enzyme causing the low fermentation yieldof the L-menthol is overcome. A YPD liquid culture medium is adopted for fermentation for 4 d, a 10% galactose aqueous solution is added in the later period from fermentation to logarithmic phase to induce expression of EEG20WW and NPPS genes and L-menthol metabolic pathway genes, normal growth and reproduction of cells are guaranteed, and the fermentation yield of the L-menthol reaches 30.14 mg / Lat most.

Description

technical field [0001] The invention relates to a recombinant yeast with improved L-menthol production, which belongs to the technical field of metabolic engineering. Background technique [0002] L-menthol (L-menthol) is a highly volatile monocyclic monoterpene substance, white needle-like crystals, easily soluble in organic solvents such as petroleum ether and chloroform, and slightly soluble in water. L-menthol is one of the most sold spices in the world, with a global annual demand of more than 20,000 tons. L-menthol is widely used in food, daily chemical, medicine, tobacco and other fields because of its cooling, sterilizing, antipruritic and analgesic effects; and it can also be used as a precursor to synthesize other fragrance substances. At present, the industrial production method of L-menthol is relatively simple, mainly natural extraction and chemical synthesis. The natural extraction method requires a large amount of mint plant raw materials, and the chemical sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/88C12N9/02C12N9/04C12N9/90C12N15/53C12N15/60C12N15/61C12P7/02C12R1/865
CPCC12N9/88C12N9/0006C12N9/0073C12N9/0081C12N9/001C12N9/90C12P7/02C12Y402/0305C12Y402/03051C12Y401/01033C12Y101/01088C12Y402/0302C12Y114/15006C12Y103/01082C12Y503/01021C12Y101/01207
Inventor 刘龙陈坚吕雪芹堵国成李江华于文文马骏房峻
Owner JIANGNAN UNIV
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