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

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.

Concomitant Production of Erythritol and β-Carotene by Engineered Yarrowia lipolytica

“…Y. lipolytica undergoes a transition to hyphal growth in response to stress, which is regulated by multiple signaling pathways, including mitogen-activated protein kinase (MAPK) and cAMP-dependent protein kinase A (PKA) pathways. , Knockout of key genes in the MAPK or PKA signaling pathways, such as Mhy1 or RAS2 , has been used to stabilize the yeast-like phenotype and block the yeast-to-hypha transition. , Mhy1 is a transcription factor that promotes the dimorphic transition in Y. lipolytica , and deletion of Mhy1 eliminates the formation of yeast filaments without affecting the stress tolerance …”

“…RAS2, encoding for a GTP binding protein, is involved in starvation response and cell morphology, and deletion of RAS2 does not affect lycopene production in Y. lipolytica . Previous studies have shown that MHY1 is a downstream signal transducer of RAS2 in regulating the dimorphic transition in Y.…”

“…31,32 Knockout of key genes in the MAPK or PKA signaling pathways, such as Mhy1 or RAS2, has been used to stabilize the yeast-like phenotype and block the yeast-to-hypha transition. 18,33 Mhy1 is a transcription factor that promotes the dimorphic transition in Y. lipolytica, and deletion of Mhy1 eliminates the formation of yeast filaments without affecting the stress tolerance. 34 RAS2, encoding for a GTP binding protein, is involved in starvation response and cell morphology, and deletion of RAS2 does not affect lycopene production in Y. lipolytica.…”

“…34 RAS2, encoding for a GTP binding protein, is involved in starvation response and cell morphology, and deletion of RAS2 does not affect lycopene production in Y. lipolytica. 33 In this study, the knockout of Mhy1 was selected to block the yeast-to-hypha transition in erythritol-producing Y. lipolytica, and it was evaluated whether this affected the production of erythritol. As shown in Figure 3E, the strain ylxs01ΔMhy1 was cultured on a YNB plate for 4 days while maintaining the yeast form.…”

Inhibition of hyphal formation together with biochar addition promotes erythritol production by Yarrowia lipolytica

“…Y. lipolytica is a dimorphic species, that is, it is able to exist in either a single-cell free-floating state or a hyphal state (Lupish et al, 2022).…”