产肌醇毕赤酵母细胞工厂的优化

Optimization of a Komagataella phaffii cell factory for producing inositol

摘要:

【背景】肌醇是一种B族维生素，广泛应用于食品、医药、饲料等领域。微生物发酵法是最具前景的肌醇生产方法，但使用大肠杆菌生产的肌醇在食品及医药领域中的使用受到限制。毕赤酵母作为生物安全菌株是工业上生产异源蛋白的良好宿主，其本身含有天然的肌醇合成途径，具有被改造成为高效生产肌醇细胞工厂的潜力。【目的】通过代谢工程改造毕赤酵母工程菌株，降低副产物的生成并提高肌醇的产量。【方法】以实验室前期构建的产肌醇毕赤酵母工程菌株为出发菌株，确定副产物阿拉伯糖醇、核糖醇和甘露糖合成相关基因。通过关键基因敲除、发酵液中葡萄糖浓度控制降低副产物的产量。通过过表达甘油转运蛋白、甘油激酶和甘油-3-磷酸脱氢酶基因实现产肌醇毕赤酵母对甘油和葡萄糖的共利用，得到重组菌Z10。经过发酵条件优化，进一步提高Z10的肌醇产量。【结果】在最优条件下，重组菌Z10的肌醇产量达到36.7 g/L，是目前酵母类细胞工厂生产肌醇的最高值，副产物总产量与出发菌株相比降低了63.1%。【结论】在毕赤酵母中建立了降低阿拉伯糖醇、核糖醇和甘露糖合成的有效策略，并通过甘油、葡萄糖共利用及相对应的发酵条件优化提高了肌醇产量，为肌醇及其他高价值生物活性物质在毕赤酵母中的高效生产提供了重要参考。

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.