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

Baixiang Zhao,Yu Zhao,Shuhui Wang,Dongguang Xiao,Aiqun Yu,Jee Loon Foo,Cuiying Zhang,Shiqi Liu,Zhihui Lu

doi:10.1186/s13068-021-02002-z

Abstract

BackgroundIn 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.ResultsIn 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.ConclusionWe 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.

Highlights

In biological cells, promoters drive gene expression by specific binding of RNA polymerase
We systematically characterized the expression of different GFPs to determine the gene that may function as an ideal reporter in Y. lipolytica Po1g KU70∆, which was the host strain for this work
We explored the structure and functional characteristics of the promoters in Y. lipolytica, and subsequently constructed a series of promoters which are stable and efficient in this study

Summary

Introduction

Promoters drive gene expression by specific binding of RNA polymerase. The common promoter elements usually include upstream activation sequences (UAS), TATA box and core promoter [1, 2]. The core promoter significantly contributes to the regulation of gene expression and is the key factor determining the promoter strength. TATA box, the recognition site of the transcription factor TATA binding protein (TBP), is one of the first kind of functional elements identified to regulate the promoter strength of the core promoter and typically located 40–120 bp upstream of the transcription start site (TSS) [3, 6]. By combining different UAS elements (UASTEF and UAS1B) in Y. lipolytica, the expression level of a constructed promoter was sevenfold higher than the wild-type promoter [2]. By exploring the synergy between various promoter elements and understanding the working mechanism of the promoter, promoters with stronger activity and a wider range of expression levels can be constructed

Objectives

Methods

Results

Conclusion