Recombinant yeast for increasing yield of L-menthol

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...

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