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Overexpression of GRAS Rhizomucor miehei lipase in Yarrowia lipolytica via optimizing promoter, gene dosage and fermentation parameters

Qinghua Zhou, Liangcheng Jiao, Yangge Qiao, Yao Wang, Li Xu, Jinyong Yan, Yunjun Yan
Journal of biotechnology 2019 v.306 pp. 16-23
GRAS substances, Rhizomucor miehei, Yarrowia lipolytica, carboxylic ester hydrolases, catalysts, culture flasks, culture media, enzyme activity, fermentation, gene dosage, gene overexpression, genes, genetic vectors, markets, pH, promoter regions, sorbitol, yeasts
Rhizomucor miehei lipase (RML), a GRAS catalyst with wide applications, was overexpressed in Yarrowia lipolytica, also a GRAS unconventional yeast, via a combined strategy, optimization for promoter, gene dosage and fermentation process. The lipase activity of the recombinant strain was first increased from 19.5 to 26.9 U/mL via codon optimization of rml gene. Subsequently, a method was developed for constructing hybrid promoters harboring different copy number of upstream activation sequences fragment (UAS1B), and the recombinant strain Po1g/hp12d-rml 25# reached 38.9 U/mL. On this basis, expression vectors with different optimized rml gene copy numbers were constructed and introduced into Y. lipolytica Po1g. The recombinant strain Po1g/hp12d-2rml 14# carrying 12 copies of UAS1B in the upstream of pLEUmin and 2 copies of rml gene obtained the highest lipase activity of 59.6 U/mL. Moreover, in optimized shaking flask culture parameters: 5% (m/v) of d-Sorbitol, 2% (v/v) inoculation density, initial pH 7.0, and 30 mL initial culture medium, the RML activity of Po1g/hp12d-2rml 14# further reached 157 U/mL after 84-h of incubation at 28 ℃. Overall, RML activity was enhanced about 8-fold compared with the initial recombinant strain via the combined strategy, which provides a consolidated basis for the large-scale production of RML in Y. lipolytica to match urgent demand of the market.