Identification and Construction of Strong Promoters in Yarrowia lipolytica Suitable for Glycerol-Based Bioprocesses

Abstract

Yarrowia lipolytica is a non-pathogenic aerobic yeast with numerous industrial biotechnology applications. The organism grows in a wide variety of media, industrial byproducts, and wastes. A need exists for molecular tools to improve heterologous protein expression and pathway reconstitution. In an effort to identify strong native promoters in glycerol-based media, six highly expressed genes were mined from public data, analyzed, and validated. The promoters from the three most highly expressed (H3, ACBP, and TMAL) were cloned upstream of the reporter mCherry in episomal and integrative vectors. Fluorescence was quantified by flow cytometry and promoter strength was benchmarked with known strong promoters (pFBA1in, pEXP1, and pTEF1in) in cells growing in glucose, glycerol, and synthetic glycerol media. The results show that pH3 > pTMAL > pACBP are very strong promoters, with pH3 exceeding all other tested promoters. Hybrid promoters were also constructed, linking the Upstream Activating Sequence 1B (UAS1B8) with H3(260) or TMAL(250) minimal promoters, and compared to the UAS1B8-TEF1(136) promoter. The new hybrid promoters exhibited far superior strength. The novel promoters were utilized to overexpress the lipase LIP2, achieving very high secretion levels. In conclusion, our research identified and characterized several strong Y. lipolytica promoters that expand the capacity to engineer Yarrowia strains and valorize industrial byproducts.

Advances and perspectives in genetic expression and operation for the oleaginous yeast Yarrowia lipolytica

“…In particular, the eukaryotic promoters are structurally complex, spanning thousands of bases from the start site and controlling the intensity and timing of gene transcription. As of now, a variety of promoters, including both inducible and constitutive ones, have been isolated and characterized in Y. lipolytica [ 11 ]. For instance, the pXPR2 promoter, induced by peptone, was isolated in 1987 [ 12 ].…”

Refactoring the architecture of a polyketide gene cluster enhances docosahexaenoic acid production in Yarrowia lipolytica through improved expression and genetic stability

“…One of the most promising alternatives to derive sustainable DHA lies in microbial production, with the oleaginous yeast Yarrowia lipolytica as one of the major production hosts [ 5 , 7 ]. This microbe has been used in industry for 70 years [ 8 ], has proven feasibility for genetic modification [ 9 – 11 ], and allows the safe manufacturing of GRAS-awarded products [ 12 ], including organic acids [ 13 ], biofuels [ 14 ], enzymes [ 15 ], and high-value chemicals for nutritional and pharmaceutical applications [ 16 ].…”