Expanding the genetic toolbox for Cutaneotrichosporon oleaginosus employing newly identified promoters and a novel antibiotic resistance marker

BMC Biotechnology

Research

Zora S. Rerop¹  Norbert Mehlmer¹  Mahmoud Masri¹  Thomas B. Brück¹  Marion Ringel¹  Nikolaus I. Stellner² 

[1] TUM School of Natural Sciences, Department of Chemistry, Werner Siemens-Chair for Synthetic Biotechnology, Technical University of Munich, Lichtenbergstr. 4, 85748, Garching, Germany;TUM School of Natural Sciences, Department of Chemistry, Werner Siemens-Chair for Synthetic Biotechnology, Technical University of Munich, Lichtenbergstr. 4, 85748, Garching, Germany;TUM CREATE Ltd, 1 Create Way, #10-02 CREATE Tower, 138602, Singapore, Singapore;

关键词: Cutaneotrichosporon oleaginosus;    Promoter;    Dominant marker;    Oleaginous yeast;    Antibiotic resistance;    Aminoglycoside 3’-phosphotransferase;    N-acetyl transferase;   

DOI  :  10.1186/s12896-023-00812-7

 received in 2023-06-09, accepted in 2023-09-08,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundCutaneotrichosporon oleaginosus is an oleaginous yeast that can produce up to 80% lipid per dry weight. Its high capacity for the biosynthesis of single cell oil makes it highly interesting for the production of engineered lipids or oleochemicals for industrial applications. However, the genetic toolbox for metabolic engineering of this non-conventional yeast has not yet been systematically expanded. Only three long endogenous promoter sequences have been used for heterologous gene expression, further three dominant and one auxotrophic marker have been established.ResultsIn this study, the structure of putative endogenous promoter sequences was analyzed based on more than 280 highly expressed genes. The identified motifs of regulatory elements and translational initiation sites were used to annotate the four endogenous putative promoter sequences D9FADp, UBIp, PPIp, and 60Sp. The promoter sequences were tested in a construct regulating the known dominant marker hygromycin B phosphotransferase. The four newly described promoters and the previously established GAPDHp successfully initiated expression of the resistance gene and PPIp was selected for further marker development. The geneticin G418 resistance (aminoglycoside 3’-phosphotransferase, APH) and the nourseothricin resistance gene N-acetyl transferase (NAT) were tested for applicability in C. oleaginosus. Both markers showed high transformation efficiency, positive rate, and were compatible for combined use in a successive and simultaneous manner.ConclusionsThe implementation of four endogenous promoters and one novel dominant resistance markers for C. oleaginosus opens up new opportunities for genetic engineering and strain development. In combination with recently developed methods for targeted genomic integration, the established toolbox allows a wide spectrum of new strategies for genetic and metabolic engineering of the industrially highly relevant yeast.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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