One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire β-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21 %, and the highest production of β-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.

[1]  A. Velayos,et al.  A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides. , 2000, European journal of biochemistry.

[2]  Zengyi Shao,et al.  DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways , 2008, Nucleic acids research.

[3]  Jean-Marc Nicaud,et al.  Yarrowia lipolytica as a model for bio-oil production. , 2009, Progress in lipid research.

[4]  K. Cho,et al.  Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome. , 2013, Journal of biotechnology.

[5]  A. Crolla,et al.  Optimization of citric acid production from Candida lipolytica Y-1095 using n-paraffin. , 2001, Journal of biotechnology.

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

[7]  M. Álvarez,et al.  Blue-light regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides , 2000, Planta.

[8]  D. G. Gibson,et al.  Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides , 2009, Nucleic acids research.

[9]  J. Nicaud,et al.  Co-expression of heterologous desaturase genes in Yarrowia lipolytica. , 2010, New biotechnology.

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

[11]  Jing Wang,et al.  High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous , 2007, Applied and Environmental Microbiology.

[12]  J. Beckerich,et al.  Yarrowia lipolytica: a model organism for protein secretion studies. , 1998, International microbiology : the official journal of the Spanish Society for Microbiology.

[13]  Hal Alper,et al.  Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach , 2011, Applied and Environmental Microbiology.

[14]  V. Cifuentes,et al.  Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous , 2013, BMC Biotechnology.

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

[16]  Seung-Pyo Hong,et al.  Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica , 2013, Nature Biotechnology.

[17]  J. Nicaud,et al.  Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains , 2012, Biotechnology Letters.

[18]  S. Zinjarde,et al.  Environmental and industrial applications of Yarrowia lipolytica , 2009, Applied Microbiology and Biotechnology.

[19]  Guang-Yuan Wang,et al.  Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109. , 2013, Biochimica et biophysica acta.

[20]  G. Barth,et al.  Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining , 2013, Current Genetics.

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