Bidirectional hybrid erythritol-inducible promoter for synthetic biology in Yarrowia lipolytica

Microbial Cell Factories

Research

Guillaume Mottet¹  Lea Vidal²  Young-Kyoung Park²  Jean-Marc Nicaud²  Esteban Lebrun³ 

[1] Large Molecules Research, Sanofi, 94400, Vitry-Sur-Seine, France;UMR1319, Team BIMLip: Integrative Biology of Microbial Lipid Metabolism, Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Domaine de Vilvert, 78350, Jouy-en-Josas, France;UMR1319, Team BIMLip: Integrative Biology of Microbial Lipid Metabolism, Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Domaine de Vilvert, 78350, Jouy-en-Josas, France;Large Molecules Research, Sanofi, 94400, Vitry-Sur-Seine, France;

关键词: Bidirectional promoter;    Inducible;    Erythritol;    Hybrid promoter;    Co-expression;    Yarrowia lipolytica;    Synthetic biology;   

DOI  :  10.1186/s12934-023-02020-6

 received in 2022-12-16, accepted in 2023-01-05,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica.ResultsThis study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter’s strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use.ConclusionsIn this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

【 授权许可】

CC BY   
© The Author(s) 2023

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