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.