Optimization of lipid production with a genome-scale model of Yarrowia lipolytica

BackgroundYarrowia lipolytica is a non-conventional yeast that is extensively investigated for its ability to excrete citrate or to accumulate large amounts of storage lipids, which is of great significance for single cell oil production. Both traits are thus of interest for basic research as well as for biotechnological applications but they typically occur simultaneously thus lowering the respective yields. Therefore, engineering of strains with high lipid content relies on novel concepts such as computational simulation to better understand the two competing processes and to eliminate citrate excretion.ResultsUsing a genome-scale model (GSM) of baker's yeast as a scaffold, we reconstructed the metabolic network of Y. lipolytica and optimized it for use in flux balance analysis (FBA), with the aim to simulate growth and lipid production phases of this yeast. We validated our model and found the predictions of the growth behavior of Y. lipolytica in excellent agreement with experimental data. Based on these data, we successfully designed a fed-batch strategy to avoid citrate excretion during the lipid production phase. Further analysis of the network suggested that the oxygen demand of Y. lipolytica is reduced upon induction of lipid synthesis. According to this finding we hypothesized that a reduced aeration rate might induce lipid accumulation. This prediction was indeed confirmed experimentally. In a fermentation combining these two strategies lipid content of the biomass was increased by 80 %, and lipid yield was improved more than four-fold, compared to standard conditions.ConclusionsGenome scale network reconstructions provide a powerful tool to predict the effects of genetic modifications and the metabolic response to environmental conditions. The high accuracy and the predictive value of a newly reconstructed GSM of Y. lipolytica to optimize growth conditions for lipid accumulation are demonstrated. Based on these findings, further strategies for engineering Y. lipolytica towards higher efficiency in single cell oil production are discussed.

[1]  Oliver Spadiut,et al.  Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway , 2012, Microbial Cell Factories.

[2]  W. Jang,et al.  High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain. , 2010, Journal of biotechnology.

[3]  C. Neuvéglise,et al.  Hexokinase--A limiting factor in lipid production from fructose in Yarrowia lipolytica. , 2014, Metabolic engineering.

[4]  De-hua Liu,et al.  Perspectives of microbial oils for biodiesel production , 2008, Applied Microbiology and Biotechnology.

[5]  B. Dujon,et al.  Genome evolution in yeasts , 2004, Nature.

[6]  David James Sherman,et al.  Génolevures: protein families and synteny among complete hemiascomycetous yeast proteomes and genomes , 2008, Nucleic Acids Res..

[7]  David E. Ruckerbauer,et al.  Nutritional requirements of the BY series of Saccharomyces cerevisiae strains for optimum growth. , 2012, FEMS yeast research.

[8]  W. A. Scheffers,et al.  A theoretical evaluation of growth yields of yeasts , 2004, Antonie van Leeuwenhoek.

[9]  Kenneth J. Kauffman,et al.  Advances in flux balance analysis. , 2003, Current opinion in biotechnology.

[10]  Jean-Marc Nicaud,et al.  Involvement of the G3P shuttle and β-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. , 2011, Metabolic engineering.

[11]  Stephen G Oliver,et al.  Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate , 2010, Biotechnology and bioengineering.

[12]  Jason A. Papin,et al.  Applications of genome-scale metabolic reconstructions , 2009, Molecular systems biology.

[13]  S. Henry,et al.  Regulation of Gene Expression through a Transcriptional Repressor that Senses Acyl-Chain Length in Membrane Phospholipids , 2014, Developmental cell.

[14]  Markus J. Herrgård,et al.  Reconstruction and validation of Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale metabolic model. , 2004, Genome research.

[15]  Colin Ratledge,et al.  The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems , 2014, Biotechnology Letters.

[16]  J. Nielsen,et al.  Metabolic Engineering of Saccharomyces cerevisiae , 2000, Microbiology and Molecular Biology Reviews.

[17]  J. Nielsen,et al.  Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose. , 2013, Metabolic engineering.

[18]  David James Sherman,et al.  A genome-scale metabolic model of the lipid-accumulating yeast Yarrowia lipolytica , 2012, BMC Systems Biology.

[19]  J. Nicaud,et al.  Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: new insights into the role of intracellular lipases and lipid body organisation. , 2013, Biochimica et biophysica acta.

[20]  J. Nicaud,et al.  Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. , 2005, FEMS yeast research.

[21]  Ronan M. T. Fleming,et al.  Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0 , 2007, Nature Protocols.

[22]  B. Palsson,et al.  Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network. , 2003, Genome research.

[23]  W. A. Scheffers,et al.  Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation , 1992, Yeast.

[24]  M. Oldiges,et al.  Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae , 2009, Applied and Environmental Microbiology.

[25]  Sudhakar Jonnalagadda,et al.  Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis , 2012, Microbial Cell Factories.

[26]  Wei Chen,et al.  Enhanced lipid accumulation in the yeast Yarrowia lipolytica by over-expression of ATP:citrate lyase from Mus musculus. , 2014, Journal of biotechnology.

[27]  Hal S Alper,et al.  Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production , 2014, Nature Communications.

[28]  Jian Chen,et al.  Enhanced α-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism. , 2012, Journal of biotechnology.

[29]  S. P. Sineoky,et al.  Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica , 2010, Biotechnology and bioengineering.

[30]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[31]  Qiang Hua,et al.  Reconstruction and In Silico Analysis of Metabolic Network for an Oleaginous Yeast, Yarrowia lipolytica , 2012, PloS one.

[32]  C. Ratledge,et al.  A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms. , 1979, Journal of general microbiology.

[33]  Gregory Stephanopoulos,et al.  Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. , 2015, Metabolic engineering.

[34]  J. Nicaud,et al.  Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization. , 2007, Fungal genetics and biology : FG & B.

[35]  Christian Jungreuthmayer,et al.  Quantitative modeling of triacylglycerol homeostasis in yeast – metabolic requirement for lipolysis to promote membrane lipid synthesis and cellular growth , 2008, The FEBS journal.

[36]  Nathan D. Price,et al.  Version 6 of the consensus yeast metabolic network refines biochemical coverage and improves model performance , 2013, Database J. Biol. Databases Curation.

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

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

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

[40]  Thomas M. Wasylenko,et al.  The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. , 2015, Metabolic engineering.

[41]  Pedro Mendes,et al.  Yeast 5 – an expanded reconstruction of the Saccharomyces cerevisiae metabolic network , 2012, BMC Systems Biology.

[42]  Jean-Marc Nicaud,et al.  Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis. , 2015, Biochimica et biophysica acta.

[43]  Jeffrey D Orth,et al.  What is flux balance analysis? , 2010, Nature Biotechnology.