Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica

In this study, we have adopted Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica. To this end, a broad set of destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen elements. Previously validated DNA modules (regulatory elements and selection markers) were adopted as the Golden Gate bricks. The system's operability was demonstrated based on synthetic pathway of carotenoid production. This technology greatly enriches a molecular biology toolbox dedicated to this industrially relevant microorganism enabling fast combinatorial cloning of complex synthetic pathways.

[1]  Xiaoying Li,et al.  Modular construction of plasmids by parallel assembly of linear vector components. , 2013, Analytical biochemistry.

[2]  J. Nicaud,et al.  Engineering Yarrowia lipolytica to produce biodiesel from raw starch , 2015, Biotechnology for Biofuels.

[3]  Lena Osterhagen,et al.  Nonconventional Yeasts In Biotechnology A Handbook , 2016 .

[4]  M. Seman,et al.  Autocloning and Amplification of LIP2 inYarrowia lipolytica , 2000, Applied and Environmental Microbiology.

[5]  Jean-Loup Faulon,et al.  PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling , 2014, Nucleic Acids Res..

[6]  Jacob Beal,et al.  CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. , 2016, ACS synthetic biology.

[7]  W. Grajek,et al.  A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica , 2013, Microbial Cell Factories.

[8]  J. Beckerich,et al.  One-step transformation of the dimorphic yeast Yarrowia lipolytica , 1997, Applied Microbiology and Biotechnology.

[9]  Praneeth Sadda,et al.  Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae , 2015, Nucleic acids research.

[10]  J. Nicaud.  Yarrowia lipolytica , 2012, Yeast.

[11]  Jean-Loup Faulon,et al.  The GoldenBricks assembly: A standardized one-shot cloning technique for complete cassette assembly , 2012 .

[12]  Shuliang Gao,et al.  One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica , 2014, Biotechnology Letters.

[13]  Jean-Marc Nicaud,et al.  Expression of invertase activity in Yarrowia lipolytica and its use as a selective marker , 1989, Current Genetics.

[14]  M. Seman,et al.  Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica , 2000, Journal of bacteriology.

[15]  H. Alper,et al.  Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. , 2014, FEMS yeast research.

[16]  Paul Freemont,et al.  EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. , 2018, Methods in molecular biology.

[17]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[18]  A. Csikász-Nagy,et al.  Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe , 2015, Open Biology.

[19]  Nicola J. Patron,et al.  A golden gate modular cloning toolbox for plants. , 2014, ACS synthetic biology.

[20]  F. Bordes,et al.  A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica. , 2007, Journal of microbiological methods.

[21]  R. Brent,et al.  Overview of Post Cohen‐Boyer Methods for Single Segment Cloning and for Multisegment DNA Assembly , 2016, Current protocols in molecular biology.

[22]  J. Nicaud,et al.  A molecular approach to optimize hIFN α2b expression and secretion in Yarrowia lipolytica , 2010, Applied Microbiology and Biotechnology.

[23]  Yizhi Cai,et al.  Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units. , 2015, ACS synthetic biology.

[24]  G. Braus,et al.  Establishing a versatile Golden Gate cloning system for genetic engineering in fungi. , 2014, Fungal genetics and biology : FG & B.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  Rodrigo Ledesma-Amaro,et al.  Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. , 2016, Progress in lipid research.

[27]  A. Steinbüchel,et al.  Yarrowia lipolytica , 2013, Microbiology Monographs.

[28]  C. Madzak.  Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering , 2015, Applied Microbiology and Biotechnology.

[29]  William C. Deloache,et al.  A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. , 2015, ACS synthetic biology.

[30]  G. Barth,et al.  Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica , 2013, Applied and Environmental Microbiology.