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Metabolic engineering of Pichia pastoris for malic acid production from methanol
Author(s) -
Guo Feng,
Dai Zhongxue,
Peng Wenfang,
Zhang Shangjie,
Zhou Jie,
Ma Jiangfeng,
Dong Weiliang,
Xin Fengxue,
Zhang Wenming,
Jiang Min
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27575
Subject(s) - pichia pastoris , metabolic engineering , biochemistry , malic acid , yeast , methanol , chemistry , pichia , isomerase , flux (metallurgy) , citric acid cycle , recombinant dna , metabolism , gene , organic chemistry , citric acid
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.