Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Abstract

Background In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

“…Construction of hybrid promoters represents a feasible solution to obtain a preferred promoter based on practical demands [ 41 , 42 ]. Here, we tried to construction of an artificial hybrid promoter based on promoter P LRA3 .…”

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“…Two separate dyes, Nile red and green fluorescence, were employed for staining of the yeast cells to validate the peroxisomal targeting ability of ePTS1. In an earlier study, it was shown that hrGFPO, encoding the green fluorescence protein, was most strongly expressed in Po1g KU70 Δ ( Zhao et al, 2021a ). The plasmid with sequence ePTS1 added after the hrGFPO protein sequence was retransformed into yeast, resulting in strain Po1g-hrGFPO-ePTS1 ( Figure 3A ).…”

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies

“…119 Other results also confirmed P TEF as the most active among the known natural promoters of Y. lipolytica. 71,111,120 Although terminators are also one of the important components of gene expression, only a few have been well characterized. Curran et al 122 constructed a panel of short synthetic terminators that can be used for modulating gene expression both in S. cerevisiae and Y. lipolytica.…”

“…Liu et al characterized and compared a series of common promoters, finding P TEF as the strongest promoter among the 15 native promoters . Other results also confirmed P TEF as the most active among the known natural promoters of Y. lipolytica . ,, Table summarizes the studies on promoter comparison.…”