PCR & Go: A Pre-installed Expression Chassis for Facile Integration of Multi-Gene Biosynthetic Pathways

Abstract

The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast

“…Nevertheless, more studies should be performed in near future to further improve the multigene pathway integration platform. For example, similar to previous studies (Qi et al, 2020; Reider Apel et al, 2017), we found that the expression level of the same expression cassette was dependent on the integration sites. The detailed mechanisms on such a context‐dependent gene expression level (X. Chen & Zhang, 2016) are yet to be explored.…”

Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae

“…S1 . Genome integration loci and the corresponding gRNA plasmids (Supplementary Table S2 ) were constructed in our previous studies 29 , 36 – 38 . For the construction of strains VSY001-VSY024, target gene expression cassettes were amplified by PCR with 40 bp homology arms to the target chromosomal loci and co-transformed with the corresponding gRNA plasmids to the Cas9 expressing yeast strains using the LiAc/SS carrier DNA/PEG method 39 .…”

Multiplex Genome Editing in Yeast by CRISPR/Cas9 – A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways

“…In another study, Qi et al utilized the highly efficient genomic loci from Apel et al and built a yeast strain containing eight well-defined cassettes in eight different loci ( Apel et al, 2017 ; Qi et al, 2021 ). Each cassette contained a unique set of promoters and terminators with a synthetic linker in the middle, resulting in a strain with eight promoter-linker-terminator cassettes in the genome ( Figure 5C ).…”

“…However, because the Cas9 cassette, promoters, and terminators were pre-integrated into the genome, the transformation only required plasmid(s) with gRNA cassettes and donor DNA genes (without promoters and terminators). This platform successfully integrated five genes in the astaxanthin biosynthetic pathway into five different loci with 69% efficiency ( Qi et al, 2021 ).…”

“…The utilization of pre-defined endogenous or synthetic target sequences has dramatically increased the maximum number of genes that can be integrated into unique loci. This is largely because the total amount of DNA transformed into yeast can be reduced due to either shorter gRNA (typically only one is required) plasmids ( Finnigan and Thorner, 2016 ; Shi et al, 2016 ; Bourgeois et al, 2018 ; Baek et al, 2021 ) or shorter donor DNAs ( Qi et al, 2021 ). Nevertheless, this approach has two major limitations.…”