1. Instruction

Yarrowia lipolytica is an oleaginous yeast with the generally recognized as safe (GRAS) status, which is well known for its superior capacity for fatty acid synthesis.[1, 2]Specifically, its distinct endogenous metabolism, broad substrate spectrum, and robustness in the fermentation production have madeY. lipolytica as a potential organism for industrial applications.[3-5] Moreover, its relatively straightforward inherited background, well-developed genetic tools, and public availability of knockout collections, such as Po1 series, make it attractive as a metabolic engineering host.[6]Currently, Y. lipolytica has been successfully engineered to produce various natural and non-natural chemicals, including 2-phenylethanol,[4] polyketides triacetic acid lactone,[7] flavonoids,[8, 9] erythritol,[10] and so forth. In addition,Y. lipolytica is characterized by a solid ability to secrete proteins, thus it also has been developed as a platform for extracellular protein production.[11]
Currently, the productive performances of strains could be optimized by rewiring the intracellular metabolic network.[12]It is worth noting that promoter engineering is an essential means to regulate and influence the timing and pattern of gene expression at the transcriptional level, further affecting the metabolic activities of microorganisms.[13, 14] Therefore, the promoter study is of great significance in metabolic engineering and synthetic biology. In Y. lipolytica , the promoter of translation elongation factor EF-1α, namely PTEF, is a strong constitutive promoter, which is widely used in the research of gene expression[9] and cell factory construction[15]. Furthermore, the promoter PTEF has also been used to construct the artificial hybrid promoters to enhance the transcription strength or endow the inducible properties[14]. For example, Blazeck et al. truncated the PTEF sequences into multiple regions to connect with the upstream activating sequences (UAS), and found that controlling the number of UAS in series within 8-16 can significantly improve the activities of hybrid promoters.[16]Besides, researchers have characterized several endogenous promoters inY. lipolytica . For example, Juretzek et al. analyzed the intensity and induction effect of endogenous promoters PG3P, PICL1, PPOT1, PPOX1, PPOX2, and PPOX5under different carbon source conditions.[17] Liu et al. characterized 22 promoters in the lipid metabolism to gain a deep understanding of lipogenesis in Y. lipolytica .[18] Although some work on the promoters has been carried out, compared with other microorganisms, such as Saccharomyces cerevisiae , Bacillus subtilis , andetc. , achievements still need to be further replenished and developed for Y. lipolytica . Moreover, the relatively few available promoters are challenging to meet the requirement of metabolic engineering for constructing microbial Y. lipolytica factories.
Herein, we screened 81 endogenous promoters in Y. lipolytica , mainly involved in carbon metabolism, amino acid metabolism, and lipid metabolism. To accurately analyze the promoter strength and avoid background interference, we used the NanoLuc luciferase reporter method.[19] As a result, 15 strong promoters, 41 medium strength promoters, and 25 weak promoters have been characterized in this study. Among them, the strongest promoter is PMnDH2 (YALI0D18964g ), 1.60-fold of the strength of the PTEF promoter, and the weakest promoter is PPHO89 (YALI0E23859g ), 0.06% of the PTEF promoter, indicating that we obtained an endogenous promoter library with the strength spanning from 0.06% to 1.60-fold of PTEF promoter. In general, our study provides a unique and available promoter library for studying Y. lipolytica cell factories, which will have great potential for industrial applications.