The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP‐CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45‐fold increase compared to the parent strain. To improve the efficiency of site‐directed gene knockout, NHEJ‐related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by‐product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose‐6‐phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value‐added chemicals from methanol.

中文翻译：

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毕赤酵母代谢工程用于从甲醇生产苹果酸。

合理设计在重新分配代谢通量以积累所需化学物质方面的应用始终受到本地监管网络的限制。在这项研究中，重组巴斯德毕赤酵母通过代谢通量的合理重新分配，从单一甲醇中生产苹果酸。在P. pastoris中系统地评估和优化了不同的苹果酸积累模块. 重组 PP-CM301 可以从葡萄糖中产生 8.55 g/L 的苹果酸，与亲本菌株相比增加了 3.45 倍。为了提高定点基因敲除的效率，NHEJ相关蛋白Ku70被破坏，同时导致异源基因沉默。因此，通过专门设计的 FRT/FLP 系统删除了与副产物生成相关的基因，成功减少了琥珀酸和乙醇的产生。此外，甲醇同化途径的一个关键节点，葡萄糖-6-磷酸异构酶被敲除以释放被困在XuMP循环中的代谢通量，最终通过氮源优化从单独的甲醇供给中积累了2.79 g/L的苹果酸。这些结果将为代谢工程提供指导和参考。P. pastoris用甲醇生产增值化学品。