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.
Highlights
There have been growing interests in using microbial cell factories to synthesize fuels, chemicals, and pharmaceutics in recent years (Nielsen and Keasling, 2016; Lian et al, 2018c; Yang et al, 2019)
We aimed to develop a “PCR & Go” system based on expression chassis for direct integration and assembly of multi-gene biosynthetic pathways into the yeast genome by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9
Using TDH3p-mCherry-CYC1t as the reporter, we achieved nearly 100% integration efficiency and the expression levels were comparably high (Supplementary Figure 1), indicating all the eight loci could be used for subsequent studies
Summary
There have been growing interests in using microbial cell factories to synthesize fuels, chemicals, and pharmaceutics in recent years (Nielsen and Keasling, 2016; Lian et al, 2018c; Yang et al, 2019). Episomal plasmids and chromosomal integration are commonly employed for heterologous expression of genes and pathways in Saccharomyces cerevisiae. While the plasmid system suffers from low genetic stability and high maintenance cost, the integration of pathway genes into the genome is preferred for biotechnological applications (Tyo et al, 2009; Da Silva and Srikrishnan, 2012). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated systems (Cas) has revolutionized the genome editing field (Hsu et al, 2014; Shalem et al, 2015; Zhang et al, 2020) and become the thumb of rule for multiplex genome engineering of yeast cell factories (DiCarlo et al, 2013; Lian et al, 2018b). Ronda et al developed the CrEdit (CRISPR/Cas mediated genome editing) system for simultaneous integration of the three pathway
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