Genome‐wide CRISPR‐Cas9 screen reveals a persistent null‐hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica

Journal Article · 21 September 2022 · Biotechnology and Bioengineering

DOI: https://doi.org/10.1002/bit.28219 · OSTI ID:1888851

Lupish, Brian ^[1]; Hall, Jordan ^[2]; Schwartz, Cory ^[2]; Ramesh, Adithya ^[2]; Morrison, Clifford ^[2]; Wheeldon, Ian  ^[3]

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1. Department of Bioengineering University of California Riverside California USA
2. Department of Chemical and Environmental Engineering University of California Riverside California USA
3. Department of Chemical and Environmental Engineering University of California Riverside California USA, Center for Industrial Biotechnology University of California Riverside California USA

Abstract Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress‐induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome‐wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null‐hyphal phenotype— ΔRAS2 , ΔRHO5 , ΔSFL1 , ΔSNF2 , and ΔPAXIP1 . Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L ⁻¹ OD ⁻¹ from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

• Research Organization: USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); Univ. of California, Riverside, CA (United States)
• Sponsoring Organization: USDOE; USDOE Office of Science (SC)
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 1888851
• Journal Information: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 12 Vol. 119; ISSN 0006-3592
• Publisher: Wiley Blackwell (John Wiley & Sons)Copyright Statement
• Country of Publication: United States
• Language: English