Engineering Yarrowia lipolytica to produce biodiesel from raw starch

Biotechnology for Biofuels

Rodrigo Ledesma-Amaro²  Thierry Dulermo¹  Jean Marc Nicaud² 

[1] AgroParisTech, UMR Micalis, Jouy-en-Josas, France

[2] Institut Micalis, INRA-AgroParisTech, UMR1319, Team BIMLip, Biologie Intégrative du Métabolisme Lipidique, CBAI, Thiverval-Grignon, 78850, France

关键词: Biodiesel;    Metabolic engineering;    Starch;    Consolidated bioprocess;    Yarrowia lipolytica;   

Others  :  1228130
DOI  :  10.1186/s13068-015-0335-7

 received in 2015-07-20, accepted in 2015-09-03,  发布年份 2015

【 摘 要 】

Background

In the last year, the worldwide concern about the abuse of fossil fuels and the seeking for alternatives sources to produce energy have found microbial oils has potential candidates for diesel substitutes. Yarrowia lipolytica has emerged as a paradigm organism for the production of bio-lipids in white biotechnology. It accumulates high amounts of lipids from glucose as sole carbon sources. Nonetheless, to lower the cost of microbial oil production and rival plant-based fuels, the use of raw and waste materials as fermentation substrate is required. Starch is one of the most abundant carbohydrates in nature and it is constituted by glucose monomers. Y. lipolytica lacks the capacity to breakdown this polymer and thus expensive enzymatic and/or physical pre-treatments are needed.

Results

In this work, we express heterologous alpha-amylase and glucoamylase enzymes in Y. lipolytica. The modified strains were able to produce and secrete high amounts of active form of both proteins in the culture media. These strains were able to grow on starch as sole carbon source and produce certain amount of lipids. Thereafter, we expressed both enzymes in an engineered strain able to overaccumulate lipids. This strain was able to produce up to 21 % of DCW as fatty acids from soluble starch, 5.7 times more than the modified strain in the wild-type background. Media optimization to increase the C/N ratio to 90 increased total lipid content up to 27 % of DCW. We also tested these strains in industrial raw starch as a proof of concept of the feasibility of the consolidated bioprocess. Lipid production from raw starch was further enhanced by the expression of a second copy of each enzyme. Finally, we determined in silico that the properties of a biodiesel produced by this strain from raw starch would fit the established standards.

Conclusions

In this work, we performed a strain engineering approach to obtain a consolidated bioprocess to directly produce biolipids from raw starch. Additionally, we proved that lipid production from starch can be enhanced by both metabolic engineering and culture condition optimization, setting up the basis for further studies.

【 授权许可】

   
2015 Ledesma-Amaro et al.

【 图 表 】

Fig.1.

Fig.2.

Fig.3.

Fig.4.

Fig.5.

Fig.6.

Fig.7.

【 参考文献 】

• [1]Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD: Microbial engineering for the production of advanced biofuels. Nature 2012, 488(7411):320-328.
• [2]Ledesma-Amaro R: Microbial oils: a customizable feedstock through metabolic engineering. Eur J Lipid Sci Technol 2015, 117(2):141-144.
• [3]van Zyl WH, Bloom M, Viktor MJ: Engineering yeasts for raw starch conversion. Appl Microbiol Biotechnol 2012, 95(6):1377-1388.
• [4]Gray KA, Zhao L, Emptage M: Bioethanol. Curr Opin Chem Biol 2006, 10(2):141-146.
• [5]Toksoy Oner E, Oliver SG, Kirdar B. Production of ethanol from starch by respiration-deficient recombinant Saccharomyces cerevisiae. Appl Environ Microbiol. 2005;71(10):6443–5. doi:10.1128/AEM.71.10.6443-6445.2005.
• [6]Aydemir E, Demirci S, Doğan A, Aytekin AO, Sahin F. Genetic modifications of Saccharomyces cerevisiae for ethanol production from starch fermentation: a review. J Bioprocess Biotech. 2014;4(180). doi:10.4172/2155-9821.1000180.
• [7]Groenewald M, Boekhout T, Neuveglise C, Gaillardin C, van Dijck PW, Wyss M: Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential. Crit Rev Microbiol 2014, 40(3):187-206.
• [8]Zinjarde SS: Food-related applications of Yarrowia lipolytica. Food Chem 2014, 152:1-10.
• [9]Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM: Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 2009, 48(6):375-387.
• [10]Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, et al.: Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat Commun. 2014, 5:3131.
• [11]Tai M, Stephanopoulos G: Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 2013, 15:1-9.
• [12]Juretzek T, Le Dall M, Mauersberger S, Gaillardin C, Barth G, Nicaud J: Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 2001, 18(2):97-113.
• [13]Madzak C: Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl Microbiol Biotechnol 2015.
• [14]Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, et al.: Genome evolution in yeasts. Nature 2004, 430(6995):35-44.
• [15]Loira N, Dulermo T, Nicaud JM, Sherman DJ: A genome-scale metabolic model of the lipid-accumulating yeast Yarrowia lipolytica. BMC Syst Biol 2012, 6:35. BioMed Central Full Text
• [16]Pan P, Hua Q: Reconstruction and in silico analysis of metabolic network for an oleaginous yeast, Yarrowia lipolytica. PLoS One 2012, 7(12):e51535.
• [17]Morin N, Cescut J, Beopoulos A, Lelandais G, Le Berre V, Uribelarrea JL, et al.: Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica. PLoS One 2011, 6(11):e27966.
• [18]Pomraning KR, Wei S, Karagiosis SA, Kim YM, Dohnalkova AC, Arey BW, et al.: Comprehensive metabolomic, lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis. PLoS One 2015, 10(4):e0123188.
• [19]Mansour S, Bailly J, Delettre J, Bonnarme P: A proteomic and transcriptomic view of amino acids catabolism in the yeast Yarrowia lipolytica. Proteomics 2009, 9(20):4714-4725.
• [20]Wasylenko TM, Ahn WS, Stephanopoulos G: The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. Metab Eng 2015.
• [21]Beopoulos A, Mrozova Z, Thevenieau F, Le Dall MT, Hapala I, Papanikolaou S, et al.: Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl Environ Microbiol 2008, 74(24):7779-7789.
• [22]Dulermo T, Nicaud JM: Involvement of the G3P shuttle and beta-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. Metab Eng 2011, 13(5):482-491.
• [23]Beopoulos A, Haddouche R, Kabran P, Dulermo T, Chardot T, Nicaud JM: Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl Microbiol Biotechnol 2012, 93(4):1523-1537.
• [24]Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson N, et al.: Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng 2015.
• [25]Wei H, Wang W, Alahuhta M, Vander Wall T, Baker JO, Taylor LE 2nd, et al.: Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing. Biotechnol Biofuels 2014, 7(1):148. BioMed Central Full Text
• [26]Celinska E, Bialas W, Borkowska M, Grajek W: Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch Yarrowia lipolytica host. Appl Microbiol Biotechnol. 2015, 99(6):2727-2739.
• [27]Park CS, Chang CC, Kim JY, Ogrydziak DM, Ryu DD: Expression, secretion, and processing of rice alpha-amylase in the yeast Yarrowia lipolytica. J Biol Chem 1997, 272(11):6876-6881.
• [28]Kwon MJ, Jorgensen TR, Nitsche BM, Arentshorst M, Park J, Ram AF, et al.: The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger. BMC Genom 2012, 13:701. BioMed Central Full Text
• [29]Muller S, Sandal T, Kamp-Hansen P, Dalboge H: Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 1998, 14(14):1267-1283.
• [30]Gasmi N, Fudalej F, Kallel H, Nicaud JM: A molecular approach to optimize hIFN alpha2b expression and secretion in Yarrowia lipolytica. Appl Microbiol Biotechnol 2011, 89(1):109-119.
• [31]Favaro L, Jooste T, Basaglia M, Rose SH, Saayman M, Gorgens JF, et al.: Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast. Appl Microbiol Biotechnol 2012, 95(4):957-968.
• [32]Kotaka A, Sahara H, Hata Y, Abe Y, Kondo A, Kato-Murai M, et al.: Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases. Biosci Biotechnol Biochem 2008, 72(5):1376-1379.
• [33]Kosugi A, Kondo A, Ueda M, Murata Y, Vaithanomsat P, Thanapase W, et al.: Production of ethanol from cassava pulp via fermentation with a surface-engineered yeast strain displaying glucoamylase. Renew Energy 2009, 34(5):1354-1358.
• [34]Tsakona S, Kopsahelis N, Chatzifragkou A, Papanikolaou S, Kookos IK, Koutinas AA: Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi. J Biotechnol 2014, 189:36-45.
• [35]Krause DR, Wood CJ, Maclean DJ: Glucoamylase (exo-1, 4-a-D-glucan glucanohydrolase, EC-3.2.1.3) is the major starch-degrading enzyme secreted by the phytopathogenic fungus Colletotrichum gloeosporioides. J Gen Microbiol 1991, 137:2463-2468.
• [36]Sun H, Zhao P, Ge X, Xia Y, Hao Z, Liu J, et al.: Recent advances in microbial raw starch degrading enzymes. Appl Biochem Biotechnol 2010, 160(4):988-1003.
• [37]Yamada R, Yamakawa S, Tanaka T, Ogino C, Fukuda H, Kondo A: Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases. Enzyme Microb Technol 2011, 48(4–5):393-396.
• [38]Yamakawa S, Yamada R, Tanaka T, Ogino C, Kondo A: Repeated batch fermentation from raw starch using a maltose transporter and amylase expressing diploid yeast strain. Appl Microbiol Biotechnol 2010, 87(1):109-115.
• [39]Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M, et al.: Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase. Appl Environ Microbiol 2004, 70(8):5037-5040.
• [40]Ratledge C: Regulation of lipid accumulation in oleaginous micro-organisms. Biochem Soc Trans 2002, 30(Pt 6):1047-1050.
• [41]Lazar Z, Dulermo T, Neuveglise C, Crutz-Le Coq AM, Nicaud JM: Hexokinase-A limiting factor in lipid production from fructose in Yarrowia lipolytica. Metab Eng 2014, 26C:89-99.
• [42]Dulermo T, Treton B, Beopoulos A, Kabran Gnankon AP, Haddouche R, Nicaud JM: 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. Biochim Biophys Acta 2013, 1831(9):1486-1495.
• [43]Thevenieau F, Beopoulos A, Desfougeres T, Sabirova J, Albertin K, Zinjarde S, et al. Uptake and assimilation of hydrophobic substrates by the oleaginous yeast Yarrowia lipolytica. In: Timmins KN, editor. Handbook of hydrocarbon and lipid microbiology, chapter 48. Berlin: Springer; 2010 (ISBN: 978-3-540-77584-3 2010).
• [44]Aguedo M, Wache Y, Mazoyer V, Sequeira-Le Grand A, Belin JM: Increased electron donor and electron acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay. J Agric Food Chem 2003, 51(10):3007-3011.
• [45]Caspeta L, Nielsen J: Economic and environmental impacts of microbial biodiesel. Nat Biotechnol 2013, 31(9):789-793.
• [46]Ramos MJ, Fernandez CM, Casas A, Rodriguez L, Perez A: Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol 2009, 100(1):261-268.
• [47]Dulermo R, Gamboa-Melendez H, Ledesma-Amaro R, Thevenieau F, Nicaud JM: Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica. Biochim Biophys Acta 2015.
• [48]Barth G, Gaillardin C: Yarrowia lipolytica. In Non conventional yeasts in biotechnology. Edited by Wolf K. Springer, Berlin; 1996:313-388.
• [49]Pignede G, Wang HJ, Fudalej F, Seman M, Gaillardin C, Nicaud JM: Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol 2000, 66(8):3283-3289.
• [50]Nicaud JM, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, et al.: Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res 2002, 2(3):371-379.
• [51]Bordes F, Fudalej F, Dossat V, Nicaud JM, Marty A: A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica. J Microbiol Methods 2007, 70(3):493-502.
• [52]Le Dall MT, Nicaud JM, Gaillardin C: Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 1994, 26(1):38-44.
• [53]Querol A, Barrio E, Huerta T, Ramon D: Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol 1992, 58(9):2948-2953.
• [54]Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM: New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 2003, 55(3):727-737.
• [55]Browse J, McCourt PJ, Somerville CR: Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 1986, 152(1):141-145.
• [56]Ledesma-Amaro R, Santos MA, Jimenez A, Revuelta JL: Tuning single-cell oil production in Ashbya gossypii by engineering the elongation and desaturation systems. Biotechnol Bioeng 2014, 111(9):1782-1791.
• [57]Wu Y, Li R, Hildebrand DF: Biosynthesis and metabolic engineering of palmitoleate production, an important contributor to human health and sustainable industry. Prog Lipid Res 2012, 51(4):340-349.
• [58]Khot M, Kamat S, Zinjarde S, Pant A, Chopade B, Ravikumar A: Single cell oil of oleaginous fungi from the tropical mangrove wetlands as a potential feedstock for biodiesel. Microb Cell Fact 2012, 11:71. BioMed Central Full Text
• [59]Knothe G: Analyzing biodiesel: standards and other methods. J Am Oil Chem Soc. 2006, 83(10):823-833.