SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae.
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.
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 223, Room 208, 2800, Kgs. Lyngby, Copenhagen, Denmark.
Katherina García
KG
Vanegas
Department of Science and Environment, Roskilde University, Universitetsvej 1, 4000, Roskilde, Denmark.
Beata Joanna
BJ
Lehka
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 223, Room 208, 2800, Kgs. Lyngby, Copenhagen, Denmark. um@bio.dtu.dk.
Uffe Hasbro
UH
Mortensen
D064113:CRISPR-Cas Systems; D005818:Genetic Engineering / Q000379:methods*; D016681:Genome, Fungal; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
CRISPR tool; Cell factory; Genome engineering; Metabolic pathway control; Saccharomyces cerevisiae