Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction

BackgroundLimonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene.ResultsTo construct the limonene synthetic pathway in Yarrowia lipolytica, two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica. Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW.ConclusionsThis is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes.

[1]  J. Beckerich,et al.  Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. , 2004, Journal of biotechnology.

[2]  P. Niederberger,et al.  Protein expression and secretion in the yeast Yarrowia lipolytica. , 2002, FEMS yeast research.

[3]  Xueli Zhang,et al.  Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. , 2013, Metabolic engineering.

[4]  James B. Y. H. Behrendorff,et al.  2,2-Diphenyl-1-picrylhydrazyl as a screening tool for recombinant monoterpene biosynthesis , 2013, Microbial Cell Factories.

[5]  Xueli Zhang,et al.  Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae , 2012, Biotechnology and bioengineering.

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

[7]  Marina Lotti,et al.  Laboratory evolution of copper tolerant yeast strains , 2012, Microbial Cell Factories.

[8]  M. Hodson,et al.  Protocols for the Production and Analysis of Isoprenoids in Bacteria and Yeast , 2015 .

[9]  Gregory Stephanopoulos,et al.  Optimization of amorphadiene synthesis in bacillus subtilis via transcriptional, translational, and media modulation. , 2013, Biotechnology and bioengineering.

[10]  Jidong Sun.  D-Limonene: safety and clinical applications. , 2007, Alternative medicine review : a journal of clinical therapeutic.

[11]  J. Keasling,et al.  Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. , 2001, Biotechnology and bioengineering.

[12]  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.

[13]  A. Makris,et al.  Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids , 2011, Microbial cell factories.

[14]  A. Makris,et al.  Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production , 2015, Microbial Cell Factories.

[15]  D. Wei,et al.  Metabolic engineering of Pichia pastoris for the production of dammarenediol-II. , 2015, Journal of biotechnology.

[16]  R. Hampton,et al.  Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae , 1997, Applied and environmental microbiology.

[17]  P. Matthews,et al.  Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase , 2000, Applied Microbiology and Biotechnology.

[18]  Christian Willrodt,et al.  Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media , 2014, Biotechnology journal.

[19]  Jules Beekwilder,et al.  Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae , 2014, Yeast.

[20]  Anthony L. Schilmiller,et al.  Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate , 2009, Proceedings of the National Academy of Sciences.

[21]  Sean X. Liu,et al.  First approach on rice bran oil extraction using limonene , 2004 .

[22]  Antia Rodriguez-Villalon,et al.  Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways. , 2008, Journal of biotechnology.

[23]  H. Orozco,et al.  Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking , 2013, Microbial Cell Factories.

[24]  J. Keasling,et al.  Engineering microbial biofuel tolerance and export using efflux pumps , 2011, Molecular systems biology.

[25]  G. Barth,et al.  Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. , 1997, FEMS microbiology reviews.

[26]  R. Croteau,et al.  4S-limonene synthase from the oil glands of spearmint (Mentha spicata). cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. , 1993, The Journal of biological chemistry.

[27]  Wang Wei.  Recent advances on limonene,a natural and active monoterpene , 2005 .

[28]  Jian Chen,et al.  Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae. , 2013, Journal of biotechnology.

[29]  S. Moineau,et al.  Bacteriophages of lactic acid bacteria and their impact on milk fermentations , 2011, Microbial cell factories.

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

[31]  Huilei Yu,et al.  Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli , 2014, Bioresources and Bioprocessing.

[32]  Jay D Keasling,et al.  Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. , 2013, Metabolic engineering.

[33]  F. Karst,et al.  Monoterpenoid biosynthesis in Saccharomyces cerevisiae. , 2007, FEMS yeast research.

[34]  Jay D Keasling,et al.  Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. , 2009, Journal of biotechnology.

[35]  Zbigniew Lazar,et al.  Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications. , 2013 .

[36]  U. Stahl,et al.  Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast , 1998, Applied Microbiology and Biotechnology.

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

[38]  N. Callewaert,et al.  Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2 , 2012, Microbial Cell Factories.