Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas9

Abstract

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.

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

“…The integration donor helper plasmids (containing the homology arms for genome integration and promoters and terminators for heterologous gene expression) and the corresponding sgRNA plasmids were constructed in our previous studies . The α-santalene biosynthetic pathway genes, including tHMG1 , IDI1 , ERG20 , and SAS , were amplified from the chromosome of the α-santalene-producing S.…”

“…A previously described CRISPR/Cas9 system was adopted for site-specific integration in K. phaffii . The pathway gene expression cassettes together with the homology arms were PCR amplified and co-transformed with the corresponding sgRNA plasmids into the Cas9-expressing strain (STE-0) .…”

“…phaffii . The pathway gene expression cassettes together with the homology arms were PCR amplified and co-transformed with the corresponding sgRNA plasmids into the Cas9-expressing strain (STE-0) . The transformation of K.…”

“…Currently, a panel of natural product biosynthetic pathways have been reconstituted in K. phaffii, including but not limited to (+)-valencene, (+)-nootkatone, − lycopene, β-carotene, astaxanthin, , betaxanthin, menaquinone-4, lovastatin, and dammarenediol-II . Nevertheless, there has been no report on the biosynthesis of α-santalene in K.…”

Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms

“…11 The discrepancy might be caused by the integration of RAD52 expression cassette into different loci (HIS4 locus in the present study and AOX1 locus in the previous report), which had been found to affect the expression level of heterologous genes significantly. 12 On the basis of the ScRAD52 overexpression strain (PpHR1), we further introduced RAD59, MRE11, and SAE2 from S. cerevisiae, leading to the construction of PpHR2, PpHR2A, and PpHR2B, respectively. As shown in Figure 2A and Supplementary Table S2, compared with PpHR1, three-loci integration efficiency using 40 bp homology arms was increased from ∼50% to ∼70%, with the highest integration efficiency achieved in PpHR2.…”