Rhodosporidium toruloides recombinant expression strain with high yield of ergothioneine and construction method and application of rhodosporidium toruloides recombinant expression strain

Abstract

The invention discloses a rhodosporidium toruloides recombinant expression strain with high yield of ergothioneine as well as a construction method and application of the rhodosporidium toruloides recombinant expression strain. The method comprises the following steps: introducing and integrating ergothioneine synthesis genes egt1 and egt2 obtained by cloning from Neurospora crassa into a rhodosporidium toruloides genome, and screening to obtain a high-yield ergothioneine strain. Compared with an original strain, the yield of ergothioneine fermented by a 5L fermentation tank of the recombinant strain is increased by 270.2% compared with that of the original strain and reaches about 8.5 g / L, and a good foundation is laid for industrial production of ergothioneine by the Rhodosporidium torulosporidium strain. Meanwhile, the fermentation period is short, fermentation products are free of toxic and side effects, the metabolic pathway transformation difficulty of the rhodotorula glutinis is small, and operability is higher.

Description

technical field [0001] The invention relates to the field of gene recombination, in particular to a recombinant expression strain of Rhodosporidium toruloides with high ergothioneine production and its construction method and application. Background technique [0002] Ergothioneine (Ergothioneine, EGT) was first isolated by Tamcet in 1909 from Claviceps purpurea, a rare natural chiral histidine-derived thiol compound, also known as 2-thio-L-histidine Acid trimethyl inner salt. It mostly exists in animals and plants but cannot be synthesized by the animal body itself, and can only be ingested from food and absorbed, accumulated, and stored in the body. At the same time, EGT, as an important active substance in the body, has various physiological functions such as scavenging free radicals, helping the body detoxify, maintaining DNA biosynthesis, normal cell growth and cellular immunity, etc., so it is also considered to be a unique, Multifunctional cell physiological protect...

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Problems solved by technology

For example, Osawa R etc. performed heterologous expression of the egt gene of Mycolicibacterium smegmatis in Escherichia coli, split and connected the genes in the gene cluster into multiple plasmids, and then transformed the plasmids into Escherichia coli, optimized Expression of recombinant enzymes to increase EGT production, shake flask fermentation EGT production reached 24mg / L; on this basis, Tanaka N et al. overexpressed cysteine, cultured in fermenters, and continuously supplemented precursors such as histidine Substance, 1.3g / L EGT was finally obtained after 216 hours of fermentation, which is currently the method with the highest yield of EGT produced by E.

Takusagawa S et al. enhanced the expression of the egt1 and egt2 genes derived from Neurospora crassa in Aspergillus oryzae, and increased the EGT production of the Aspergillus oryzae host to a level of 231mg / L. However, the fermentation cycle of Aspergillus oryzae was as long as 10 Days or more, the yield of ergothioneine is low, and subsequent transformation of its metabolic pathway is difficult

Steven A et al. used Saccharomyces cerevisiae to heterologously express EGT synthase genes (egtA, egtB, egtC, egtD, egtE) from multiple sources such as ergot and Mycobacterium smegmatis, and feed batch fermentation in a 1L fermenter. Under the same conditions, the EGT yield of 0.598g / L was obtained after 84 hours of cultivation, but in the tank scale-up test, the strain showed obvious growth inhibition and premature death, and the yield did not increase

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