Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose

Abstract

The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica is developed and is used it to activate native β-glucosidase expression to support growth on cellobiose. A series of different transcriptional activators are compared for their effectiveness in Y. lipolytica, with the synthetic tripartite activator VPR yielding the highest activation. A VPR-dCas9 fusion is then targeted to various locations in a synthetic promoter driving hrGFP expression, and activation is achieved. Subsequently, the CRISPRa system is used to activate transcription of two different native β-glucosidase genes, facilitating enhanced growth on cellobiose as the sole carbon source. This work expands the synthetic biology toolbox for metabolic engineering in Y. lipolytica and demonstrates how the programmability of the CRISPR-Cas9 system can enable facile investigation of transcriptionally silent regions of the genome.

Multiplexed CRISPR technologies for gene editing and transcriptional regulation

“…4f). Multiplexed CRISPR-Cas technologies are frequently used to simultaneously activate and repress multiple genes at once, an approach that has been used to enhance cellobiose consumption in Yarrowia lipolytica, isoprenoid production in S. cerevisiae, and n-butanol and terpenoid production in E. coli [88][89][90][91] . More recently, a study increased succinic acid production approximately 150-fold in E. coli by co-expressing an inducible dCas9 with 20 sgRNAs to knockdown 6 genes 48 .…”

Synthetic biology tools for engineering Yarrowia lipolytica

“…dCas9 is fused with a transcription factor and targets the upstream region of the gene, delivering the transcription factor to the promoter; this process enhances transcription efficiency. The abbreviations are as follows: gDNA: genomic DNA; sgRNA: single-guide RNA; dCas9: catalytically inactive Cas9; RNAP: RNA polymerase; TF: transcription factor; Mxi1: repressor; and VPR: synthetic activator domain ( Schwartz et al, 2018 ). …”

Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system

“…As the first CRISPR research in Y. lipolytica by Schwartz et al ( 2016a ), both expression of the active Cas9 protein in the CRISPR/Cas9 system and dCas9 in CRISPRi and CRISPRa systems after that has been engineered using a strong constitutive promoter as well as a SV40 nuclear localization signal (Gao et al 2016 ; Schwartz et al 2017 ; Schwartz et al 2018 ; Schwartz and Wheeldon 2018 ; Holkenbrink et al 2018 ; Zhang et al 2018 ). There are two general strategies for the expression of Cas9/dCas9—one based on plasmids and the other on chromosomal integration.…”