Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

Automated summary

In this study, an engineered Pichia pastoris strain was constructed for the production of Triacetic acid lactone (TAL). The TAL biosynthetic pathway was introduced by integrating a 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). The rate-limiting step of TAL synthesis was removed by introducing a mutant gene for acetyl-CoA carboxylase from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Additionally, the phosphoketolase/phosphotransacetylase pathway (PK pathway) was introduced to enhance intracellular acetyl-CoA supply.