SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae

TLDR

Saccharomyces cerevisiae is increasingly used as a cell factory, but construction time remains a major obstacle to bio‑production. The study aims to develop tools that accelerate yeast cell factory construction. SWITCH uses Cas9/dCas9 to alternate between genetic engineering and pathway control states, with the TAPE technique optimizing protospacer efficiency for recombination. SWITCH enabled the rapid insertion of five genes for naringenin production and, by downregulating TSC13 in the pathway control state, increased product yield while reducing by‑product formation.

Abstract

Background The yeast Saccharomyces cerevisiae is increasingly used as a cell factory. However, cell factory construction time is a major obstacle towards using yeast for bio-production. Hence, tools to speed up cell factory construction are desirable. Results In this study, we have developed a new Cas9/dCas9 based system, SWITCH, which allows Saccharomyces cerevisiae strains to iteratively alternate between a genetic engineering state and a pathway control state. Since Cas9 induced recombination events are crucial for SWITCH efficiency, we first developed a technique TAPE, which we have successfully used to address protospacer efficiency. As proof of concept of the use of SWITCH in cell factory construction, we have exploited the genetic engineering state of a SWITCH strain to insert the five genes necessary for naringenin production. Next, the naringenin cell factory was switched to the pathway control state where production was optimized by downregulating an essential gene TSC13, hence, reducing formation of a byproduct. Conclusions We have successfully integrated two CRISPR tools, one for genetic engineering and one for pathway control, into one system and successfully used it for cell factory construction.