Development of metabolic engineering strategies in Komagataella phaffii for the reduction of cellular methanol requirements based on the cellular response to non-repressive compounds of the pAOX1 promoter

The methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) is widely recognized as a prominent platform for recombinant protein (rProt) production. This yeast is capable of oxidizing methanol to support energy production and biomass formation. In this non-conventional yeast, the methanol-regulated pAOX1 promoter has been widely used to drive the expression of recombinant proteins. However, the use of methanol raises significant safety concerns due to its high flammability, particularly in large-scale industrial applications. Consequently, the main objective of this thesis was to explore alternatives to reduce or eliminate the reliance on methanol.
In this framework, disruption of the formate dehydrogenase gene (FDH1) was employed as a strategy to modify the pAOX1 expression system. This genome edition successfully converted the system into a self-inducing platform for rProt synthesis, eliminating the need for methanol or other inducers to trigger gene expression. Further investigation revealed that this induction mechanism is mediated by cytoplasmic formate generated from THF-C1 metabolism, which cannot be converted to carbon dioxide in an FDH1 knockout strain. Notably, this discovery showed that the pAOX1 promoter could be induced under non-repressive conditions, such as in the presence of sorbitol. The efficacy of this system was demonstrated through the synthesis of two model proteins: intracellular eGFP and secreted CalB lipase from Candida antarctica.
Further into this investigation, metabolic engineering efforts focused on optimizing sorbitol uptake to enhance cell growth and rProt production in the FDH1 knockout strain. Sorbitol metabolism was identified as the bottleneck, and overexpression of the sorbitol dehydrogenase gene (SOR1) significantly improved the growth in K. phaffii, leading to a 2.5-fold increase in specific cell growth rate. Despite these improvements, an unexpected impairment in pAOX1 induction was observed. This study proposes several hypotheses to explain this issue, which are discussed in detail in this work.
Overall, this investigation lays the groundwork for a deeper understanding of pAOX1 induction and highlights new avenues for exploring the underlying regulatory mechanisms. These insights will be pivotal in optimizing the system's potential for advancing biotechnological applications in this innovative methanol-free platform.