View metadata,Downloaded citation and from similar orbit.dtu.dk papers on:at core.ac.uk Nov 09, 2017 brought to you by CORE provided by Online Research Database In Technology Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica Kerkhoven, Eduard J.; Pomraning, Kyle R.; Baker, Scott E.; Nielsen, Jens Published in: n p j Systems Biology and Applications Link to article, DOI: 10.1038/npjsba.2016.5 Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Kerkhoven, E. J., Pomraning, K. R., Baker, S. E., & Nielsen, J. (2016). Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica. n p j Systems Biology and Applications, 2, [16005]. DOI: 10.1038/npjsba.2016.5 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. www.nature.com/npjsba All rights reserved 2056-7189/16 ARTICLE OPEN Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica Eduard J Kerkhoven1, Kyle R Pomraning2, Scott E Baker2 and Jens Nielsen1,3 Yarrowia lipolytica is a promising microbial cell factory for the production of lipids to be used as fuels and chemicals, but there are few studies on regulation of its metabolism. Here we performed the first integrated data analysis of Y. lipolytica grown in carbon and nitrogen limited chemostat cultures. We first reconstructed a genome-scale metabolic model and used this for integrative analysis of multilevel omics data. Metabolite profiling and lipidomics was used to quantify the cellular physiology, while regulatory changes were measured using RNAseq. Analysis of the data showed that lipid accumulation in Y. lipolytica does not involve transcriptional regulation of lipid metabolism but is associated with regulation of amino-acid biosynthesis, resulting in redirection of carbon flux during nitrogen limitation from amino acids to lipids. Lipid accumulation in Y. lipolytica at nitrogen limitation is similar to the overflow metabolism observed in many other microorganisms, e.g. ethanol production by Sacchromyces cerevisiae at nitrogen limitation. npj Systems Biology and Applications (2016) 2, 16005; doi:10.1038/npjsba.2016.5; published online 3 March 2016 INTRODUCTION interactions. For this purpose we generated a comprehensive The yeast Yarrowia lipolytica has a high potential as microbial cell genome-scale metabolic model (GEM) of Y. lipolytica metabolism. factory for the production of biofuels and chemicals. Y. lipolytica is Although three GEMs of Y. lipolytica have been published 10 11 12 an oleaginous yeast, capable of accumulating over 70% of its previously (iYL619_PCP, iNL895 and iMK735 ), our model is biomass as lipids.1 These lipids are stored in lipid bodies and exist far more comprehensive, based on the more recent Yeast 7.11 13 primarily of triacylglycerols (TAGs) with different chain lengths, consensus network and curated to include unique reactions which can function as intermediates for the production of from both iYL619_PCP and iNL895. Further curation was advanced biofuels.2 Studying metabolism in Y. lipolytica is of performed with available literature data and improved annotation interest as its dysregulation allows engineering opportunity for of the Y. lipolytica genome as described in Supplementary increased lipid production. As an example, a push-and-pull Informations 1 and 2. The resulting model is the most genetic engineering strategy has been employed to divert the comprehensive GEM of an oleaginous yeast to date, and provides carbon flux during nitrogen restriction towards TAG production.3 the biofuel research community with a tool for further identifying Overexpression of acetyl-CoA carboxylase (ACC1) pulls carbons engineering targets and a framework to unravel regulation of from the typical excretion metabolite citrate into fatty acid metabolism. We name this model iYali4, as the fourth published biosynthesis, while overexpression of diacylglycerol acyltransfer- GEM of Y. lipolytica. ase (DGA1) pushes the fatty acids into the TAGs. This approach has been successful as almost all carbons are diverted from citrate Physiological characterisation of chemostat cultures excretion into TAG biosynthesis. However, while efforts have been made to increase lipid production in Y. lipolytica,4 currently our A high-lipid producing strain of Y. lipolytica, overexpressing knowledge of how lipid accumulation is regulated in Y. lipolytica is diacylglycerol acyltransferase (DGA1), the last step of TAG limited, whereas regulators as SNF1,5 MIG16 and MGA27 biosynthesis, was cultivated in a bioreactor under chemostat have been shown to affect lipid accumulation. Systems level conditions. Restrictive availability of ammonium (as nitrogen analysis is an excellent tool for probing regulatory mechanisms, source) was compared to the restrictive availability of glucose as demonstrated extensively for Sacchromyces cerevisiae.8,9 (as carbon source), all at the same dilution rate (0.05 per h). – However no such approach has been applied to Y. lipolytica to Carbon nitrogen ratios of 2.2 and 110 were selected as date, and we therefore undertook the first integrated analysis of representing carbon versus nitrogen limitation (Supplementary fi lipid accumulation in Y. lipolytica. Information 3). When the cultures reached steady state, de ned at a constant OD600 and O2 partial pressure in the exhaust gas, RESULTS samples were taken for measurements (Figure 1). An overview of the fermentation profile shows that the specific Reconstruction of genome-scale metabolic model glucose consumption rate (qgluc) was similar at both nitrogen Integration of multilevel data requires a framework that can and carbon limitations, and also the biomass yield (YSX) was accommodate different data types and allows for mapping of comparable (Table 1). This is in stark contrast with for instance 1Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden; 2Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA and 3Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. Correspondence: J Nielsen ([email protected]) Received 14 September 2015; revised 23 November 2015; accepted 7 December 2015 © 2016 The Systems Biology Institute/Macmillan Publishers Limited Regulating lipid accumulation in Yarrowia EJ Kerkhoven et al 2 Lipid composition Total lipid content HPLC Update iYali4 250 fluxes PI N-lim TM 200 LC-CAD and C-lim –1 3x biomass C-lim PS 150 GC-MS in GEM THE RAVEN PC 100 Annotate TOOLBOX 50 mg gDCW RNAseq new genes 3x PE 0 N-lim DE analysis Random sampling CL Total fatty acid composition Differentially Potential Differentially PA 16:0 expressed transcriptional changed Lipid classes genes regulation fluxes ES 16:1 Figure 1. Experimental design. Triplicate chemostats were run at FFA 18:0 steady-state, after which various samples were taken. Strain-specific TAG 18:1 GEMs were generated using the experimental data and the Fatty acid chain predicted flux changes were compared with differentially expressed SE 18:2 transcripts, indicating potential transcriptional regulation. 020406080 40% 20% 0% mg gDCW–1 Table 1. Physiological parameters Figure 2. Lipid and total fatty acid composition. Error bars are s.d. of three independent biological replicates. CL, cardiolipin; ES, Carbon Nitrogen restriction ergosterol; FFA, free fatty acid; PA, phosphatidate; PC, phosphati- restriction dyl-choline; PE, phosphatidylethanolamine; PI, phosphatidyl-inositol; PS, phosphatidyl-serine; SE, steryl ester; TAG, triacylglycerol. Specific growth rate (per hour) 0.047 (±0.004) 0.048 (±0.002) Biomass concentration (g/l) 2.1 (±0.1) 2.7 (±0.5) Increases in lipids during nitrogen restriction ± Extracellular glucose 0 17.9 ( 0.8) The lipid compositions of Y. lipolytica were measured for both concentration (g/l) nutrient restrictions, and the most dominant phospholipids were qGluc (mmol gDW/h) 0.61 (±0.06) 0.64 (±0.06) q (mmol gDW/h) 1.3 (±0.3) 2.1 (±0.3) found to be PE, PC and PI (Figure 2), corroborating previous O2 23 qCO2 (mmol gDW/h) 1.5 (±0.1) 2.2 (±0.3) measurements of Y. lipolytica. The low steryl ester (SE) content RQ (− ) 1.15 ( ±0.20) 1.0 (±0.1) was surprising, as SE is typically identified as a storage lipid ± ± YSX (gDW g/glucose) 0.43 ( 0.02) 0.42 ( 0.01) together with TAG, and lipid droplets normally contain 7.8–14% Dissolved oxygen (%) 82 (±4) 74 (±10) steryl esters.24 However, other reports state that steryl esters are 25 Abbreviation: RQ, respiratory quotient. only present in very small amounts. Data are means (s.d.) from three independent chemostats. The total lipid content increased from 40.5 mg/gDW (±5.7 mg/gDW)