Development of a production strategy based on the modification of metabolic regulation processes in Pichia pastoris.

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Chinese explanation / 中文解读

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Original abstract

Komagataella phaffii is one of the most widely used yeast cell factories for recombinant protein (rProt) production in the industry. Its success has been largely attributed to the strong alcohol oxidase 1 promoter (pAOX1), which is traditionally induced by methanol. However, the toxicity and handling constraints of methanol limit its industrial applicability. Recently, formate dehydrogenase (FDH) deficient strain of K. phaffii has been reported that formate as a key metabolite for pAOX1 induction in sorbitol media. This doctoral research has been focused on developing methanol-free induction strategies for pAOX1 in this FDH-deficient strain, to improve rProt production. These results showed that sorbitol dehydrogenase (SOR1) overexpression gene, based on the slow sorbitol uptake limitation, enhanced sorbitol metabolism and growth rate. However, it also decreased intracellular formate accumulation, a key metabolite involved in pAOX1 induction in this strain. Thereby, this effect reduced pAOX1 induction and the synthesis of model rProt, such as enhanced green fluorescent protein (eGFP) and Candida antarctica lipase (CalB). Oxygen transfer conditions (OTC) were also found to play a critical role in pAOX1 regulation, formate accumulation, and increased eGFP production. Furthermore, engineering of the L-serine biosynthesis pathway through overexpression of 3-phosphoglycerate dehydrogenase (SER3) gene, based on previous results obtained in serine-sorbitol media, enhanced intracellular formate levels and improved the balance between growth and rProt production, SER3 strains exhibited up to 48% higher specific eGFP fluorescence and 30% higher glucose oxidase (Gox) activity compared to the parental strain. These findings highlight serine metabolism as a potential contributor to the link between central carbon metabolism and pAOX1 induction. In addition, signal peptide engineering was investigated as an independent approach to improve recombinant protein secretion efficiency based on different composition of pre- and pro-regions of signal peptides (SPs) of Yarrowia lipolytica. Yps3Pre-Lip2Pro construct yielded the highest laccase activity, highlighting the importance of secretion signal composition and design in overall rProt production performance and secretion efficiency. These findings expand current knowledge on the molecular mechanisms underlying pAOX1 activation and provide a foundation for designing safer, more sustainable, and efficient recombinant protein production systems in K. phaffii.

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