Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Komagataella phaffii GS115 is a proven heterologous expression system and has recently been exploited for the production of value‐added biochemicals from glucose through metabolic engineering. A major challenge for high‐level biochemical production is the appropriate distribution of carbon flux between cell growth and product biosynthesis. In this study, we report the development of a synergetic glucose and glycerol coutilization strategy for K. phaffii, potentially enabling this strain to consume glycerol for growth while conserving more glucose for product formation. First, several potential genes encoding mediator proteins and transcriptional factors that were considered to be associated with carbon catabolite repression in K. phaffii were screened, and deletion of gss1, a glucose sensor, appeared to be able to eliminate the glucose‐induced repression of glycerol utilization in a mixed glucose–glycerol medium. Transcriptome comparisons between the parent strain and the Δgss1 mutant were then performed, and the glycerol‐metabolism genes that were subjected to glucose regulation were identified. Second, coutilization of glucose and glycerol in K. phaffii was achieved by overexpressing genes relevant to glycerol metabolism, namely, gt1, gut1, and gut2. Furthermore, knockout or knockdown of pfk and zwf genes resulted in a reduction of carbon flux from glucose towards glycolysis and the pentose phosphate pathway. With these efforts, the cell metabolism of the final strain was divided into growth and production modules. This study describes a promising strategy to address the challenge of carbon flux distribution in K. phaffii, and would be valuable in engineering this strain as a versatile fermentation platform for biochemical production.