Guo Feng, Dai Zhongxue, Peng Wenfang, Zhang Shangjie, Zhou Jie, Ma Jiangfeng, Dong Weiliang, Xin Fengxue, Zhang Wenming, Jiang Min
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Science, Hubei University, Wuhan, China.
Biotechnol Bioeng. 2021 Jan;118(1):357-371. doi: 10.1002/bit.27575. Epub 2020 Oct 7.
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.
合理设计在重新分配代谢通量以积累所需化学品方面的应用总是受到天然调控网络的限制。在本研究中，通过合理重新分配代谢通量构建了重组毕赤酵母，用于从单一甲醇生产苹果酸。在毕赤酵母中系统评估并优化了不同的苹果酸积累模块。重组菌株PP-CM301以葡萄糖为底物可生产8.55 g/L苹果酸，与亲本菌株相比提高了3.45倍。为提高定点基因敲除效率，破坏了与非同源末端连接（NHEJ）相关的蛋白Ku70，但导致了异源基因的沉默。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，成功降低了琥珀酸和乙醇的产量。此外，敲除甲醇同化途径中的一个关键节点——葡萄糖-6-磷酸异构酶，以释放被困在木酮糖单磷酸（XuMP）循环中的代谢通量，最终通过氮源优化实现了从单一甲醇进料中积累2.79 g/L苹果酸。这些结果将为毕赤酵母代谢工程从甲醇生产增值化学品提供指导和参考。
