This invention discloses a method for constructing a high-
succinic acid-producing
Pichia pastoris
cell factory, using
Pichia pastoris (… Komagataella phaffii Using GS115 as the
chassis, a reductive TCA cycle enhancement strategy and an sdh5 knockout strategy were first constructed, respectively. It was found that a single strategy was insufficient to effectively achieve
succinic acid accumulation. Further, the two strategies were combined to obtain the engineered strain SAOG, which achieved
succinic acid accumulation. Based on this, the malate
transporter SpMAE was introduced to enhance the transmembrane transport capacity of succinic acid, and
histidine auxotrophic
recovery was combined to improve the growth and
fermentation performance of the strain, resulting in the engineered strain SH, whose succinic acid accumulation was 25.1% higher than that of SAOG. Finally, the engineered strain SH achieved a succinic acid yield of 10.47 g / L in shake-flask
fermentation and 112.6 g / L after 120 h of continuous fed-
batch fermentation in a 5 L fermenter. This invention achieves efficient succinic acid synthesis in the
Pichia pastoris
chassis through the synergistic regulation of reductive TCA pathway enhancement, succinic
acid oxidation blocking, enhanced transmembrane transport, and auxotrophic
recovery, exhibiting stable
fermentation performance, high yield, and good industrial application potential.