Optimization of a Komagataella phaffii cell factory for producing inositol

Abstract:

[Background] Inositol is a member of the vitamin B group and has been widely used in the fields of food, medicine, and feed. Microbial fermentation is considered as the most promising method for inositol production, while the inositol produced by Escherichia coli is restricted for food and medicine uses. As a GRAS (generally recognized as safe) strain, Komagataella phaffii has been employed as a robust host for producing heterologous proteins. K. phaffii possesses a native synthetic pathway of inositol and thus has great potential to be modified as a cell factory for the efficient production of inositol.[Objective] To reduce the by-products and enhance the inositol production via metabolic engineering of K. phaffii. [Methods] With the inositol-producing K. phaffii strain which was previously constructed in our lab as the starting strain, we identified the genes involved in the synthesis of arabitol, ribitol, and mannose. Through deletion of the key genes for synthesis and control of the glucose concentration in the fermentation, we decreased the production of by-products. The co-utilization of glycerol and glucose was achieved in the inositol-producing K. phaffii strain by overexpression of glycerol transporter, glycerol kinase, and glycerol-3-phosphate dehydrogenase, and a recombinant strain Z10 was obtained. Furthermore, inositol production was improved by optimization of the fermentation conditions. [Results] The inositol production of the recombinant strain Z10 under the optimal conditions reached 36.7 g/L, the highest titer reported to date by a yeast cell factory. The by-products of Z10 was reduced by 63.1% compared with that of the starting strain. [Conclusion] An effective strategy was established in K. phaffii for decreasing the production of arabitol, ribitol, and mannose. Through the co-utilization of glycerol and glucose and the optimization of fermentation conditions, the inositol production was increased. The findings of this study provide a reference to the application of K. phaffii for the efficient production of inositol and other high-value bioactive substances.