Pichia pastoris, an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris. First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, β-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.

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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.