CRISPR interference technology for development of more tolerant industrial yeast strains (Milan)

Second generation bioethanol using lignocellulosic biomass as raw material is a promising alternative to bioethanol produced from sugar-based feedstocks. In addition to sugars, lignocellulosic hydrolysates also contain inhibitors that impair microbial growth. One way to tackle the low productivities is to develop new strains with increased tolerance towards inhibitors.Over the past few years, different CRISPR technologies have been developed to accelerate the construction of new strains. The CRISPR interference (CRISPRi) technology utilizes a catalytically inactive Cas9 (dCas9) to modulate the expression of genes targeted by a sgRNA, allowing the alteration of essential genes and the manipulation of multiple traits without altering the target sequence.In the present work, our goal was to use CRISPRi to improve the inhibitor tolerance of a polyploid industrial yeast strain. We explored different strategies to overcome the challenges of implementing CRISPRi in an industrial strain. As a proof of concept, the expression of a gene encoding a fluorescent protein was modulated using dCas9 with different activation or repression domains. Changes in fluorescence were measured by flow cytometry and changes in expression were verified by qPCR, validating the use of CRISPRi for alteration of gene expression in an industrial yeast strain.Subsequently, a number of genes previously identified to be involved in inhibitor tolerance were selected as targets for CRISPRi. The performance of the novel strains during growth in the presence of different inhibitors was analysed in a high-throughput platform, leading to identification of strains where the altered gene expression led to improved tolerance.This work shows that the CRISPRi technology can be used to accelerate the development of more robust, industrial production hosts