Abstract
In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica.
Graphical Abstract
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Amadio J, Casey E, Murphy CD (2013) Filamentous fungal biofilm for production of human drug metabolites. Appl Microbiol Biotechnol 97:5955–5963. https://doi.org/10.1007/s00253-013-4833-x
Bankar AV, Kumar AR, Zinjarde SS (2009) Environmental and industrial applications of Yarrowia lipolytica. Appl Microbiol Biotechnol 84:847–865. https://doi.org/10.1007/s00253-009-2156-8
Barth G, Gaillardin C (1996) Yarrowia lipolytica. In: Wolf K (ed) Nonconventional yeasts in biotechnology. Springer Berlin Heidelberg, pp 313–388. https://doi.org/10.1007/978-3-642-79856-6_10
Beopoulos A, Nicaud J-M, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206. https://doi.org/10.1007/s00253-011-3212-8
Blazeck J, Hill A, Jamoussi M, Pan A, Miller J, Alper HS (2015) Metabolic engineering of Yarrowia lipolytica for itaconic acid production. Metab Eng 32:66–73. https://doi.org/10.1016/j.ymben.2015.09.005
Braga A, Belo I (2013) Immobilization of Yarrowia lipolytica for aroma production from castor oil. Appl Biochem Biotechnol 169:2202–2211. https://doi.org/10.1007/s12010-013-0131-4
Briffaud J, Engasser J (1979) Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor. Biotechnol Bioeng 21:2083–2092. https://doi.org/10.1002/bit.260211113
Carly F, Steels S, Telek S, Vandermies M, Nicaud J-M, Fickers P (2018) Identification and characterization of EYD1, encoding an erythritol dehydrogenase in Yarrowia lipolytica and its application to bioconvert erythritol into erythrulose. Bioresour Technol 247:963–969. https://doi.org/10.1016/j.biortech.2017.09.168
Carly F, Vandermies M, Telek S, Steels S, Thomas S, Nicaud J-M, Fickers P (2017) Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering. Metab. Eng. 42:19–24. https://doi.org/10.1016/j.ymben.2017.05.002
Cervantes-Chávez JA, Kronberg F, Passeron S, Ruiz-Herrera J (2009) Regulatory role of the PKA pathway in dimorphism and mating in Yarrowia lipolytica. Fungal Genet Biol 46:390–399. https://doi.org/10.1016/j.fgb.2009.02.005
Cervantes-Chávez JA, Ruiz-Herrera J (2006) STE11 disruption reveals the central role of a MAPK pathway in dimorphism and mating in Yarrowia lipolytica. FEMS Yeast Res 6:801–815. https://doi.org/10.1111/j.1567-1364.2006.00084.x
Escamilla-García E, O’Riordan S, Gomes N, Aguedo M, Belo I, Teixeira J, Belin J-M, Waché Y (2014) An air-lift biofilm reactor for the production of γ-decalactones by Yarrowia lipolytica. Process Biochem 49:1377–1382. https://doi.org/10.1016/j.procbio.2014.05.011
Fickers P, Benetti P, Wache Y, Marty A, Mauersberger S, Smit M, Nicaud J-M (2005) Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res 5:527–543. https://doi.org/10.1016/j.femsyr.2004.09.004
Fickers P, Le Dall M-T, Gaillardin C, Thonart P, Nicaud J-M (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55:727–737. https://doi.org/10.1016/j.mimet.2003.07.003
Finnigan GC, Sterling SM, Duvalyan A, Liao EN, Sargsyan A, Garcia G, Nogales E, Thorner J (2016) Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae. Mol Biol Cell 27:2213–2233. https://doi.org/10.1091/mbc.E16-03-0177
Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, Joe Shaw A, South CR, Stephanopoulos G, Brevnova EE (2016) Engineering of a high lipid producing Yarrowia lipolytica strain. Biotechnol Biofuels 9:77. https://doi.org/10.1186/s13068-016-0492-3
Gao C, Yang X, Wang H, Rivero CP, Li C, Cui Z, Qi Q, Lin CSK (2016) Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica. Biotechnol Biofuels 9:179. https://doi.org/10.1186/s13068-016-0597-8
González-López CI, Ortiz-Castellanos L, Ruiz-Herrera J (2006) The ambient pH response rim pathway in Yarrowia lipolytica: identification of YlRIM9 and characterization of its role in dimorphism. Curr Microbiol 53:8–12. https://doi.org/10.1007/s00284-005-0070-6
Gross R, Hauer B, Otto K, Schmid A (2007) Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnol Bioeng 98:1123–1134. https://doi.org/10.1002/bit.21547
Guevara-Olvera L, Calvo-Mendez C, Ruiz-Herrera J (1993) The role of polyamine metabolism in dimorphism of Yarrowia lipolytica. Microbiology 139:485–493
Gutiérrez-Correa M, Ludeña Y, Ramage G, Villena GK (2012) Recent advances on filamentous fungal biofilms for industrial uses. Appl Biochem Biotechnol 167:1235–1253. https://doi.org/10.1007/s12010-012-9555-5
Iqbal M, Saeed A (2005) Novel method for cell immobilization and its application for production of organic acid. Lett Appl Microbiol 40:178–182. https://doi.org/10.1111/j.1472-765X.2004.01646.x
Kang H, Tsygankov D, Lew DJ (2016) Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1. Mol Biol Cell 27:1764–1775. https://doi.org/10.1091/mbc.E16-01-0014
Khalesi M, Zune Q, Telek S, Riveros-Galan D, Verachtert H, Toye D, Gebruers K, Derdelinckx G, Delvigne F (2014) Fungal biofilm reactor improves the productivity of hydrophobin HFBII. Biochem Eng J 88:171–178. https://doi.org/10.1016/j.bej.2014.05.001
Le Dall M-T, Nicaud J-M, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44
Ledesma-Amaro R, Nicaud J-M (2016) Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Prog Lipid Res 61:40–50. https://doi.org/10.1016/j.plipres.2015.12.001
Lengeler KB, Davidson RC, D’souza C, Harashima T, Shen W-C, Wang P, Pan X, Waugh M, Heitman J (2000) Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev 64:746–785
Li C, Yang X, Gao S, Wang H, Lin CSK (2017) High efficiency succinic acid production from glycerol via in situ fibrous bed bioreactor with an engineered Yarrowia lipolytica. Bioresour Technol 225:9–16. https://doi.org/10.1016/j.biortech.2016.11.016
Li M, Li Y-Q, Zhao X-F, Gao X-D (2014) Roles of the three Ras proteins in the regulation of dimorphic transition in the yeast Yarrowia lipolytica. FEMS Yeast Res 14:451–463. https://doi.org/10.1111/1567-1364.12129
Liesche J, Marek M, Günther-Pomorski T (2015) Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells. Front Microbiol 6. https://doi.org/10.3389/fmicb.2015.00107
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Madzak C (2015) Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl Microbiol Biotechnol 99:4559–4577. https://doi.org/10.1007/s00253-015-6624-z
Mauersberger S, Wang H-J, Gaillardin C, Barth G, Nicaud J-M (2001) Insertional mutagenesis in the n-alkane-assimilating yeast Yarrowia lipolytica: generation of tagged mutations in genes involved in hydrophobic substrate utilization. J Bacteriol 183:5102–5109. https://doi.org/10.1128/JB.183.17.5102-5109.2001
Mitra S, Banerjee P, Gachhui R, Mukherjee J (2011) Cellulase and xylanase activity in relation to biofilm formation by two intertidal filamentous fungi in a novel polymethylmethacrylate conico-cylindrical flask. Bioprocess Biosyst Eng 34:1087–1101. https://doi.org/10.1007/s00449-011-0559-2
Nemati M, Webb C (2011) 2.24—immobilized cell bioreactors. In: Moo-Young M (ed) Comprehensive Biotechnology,2nd ed. Academic Press, Burlington, pp 331–346. https://doi.org/10.1016/B978-0-08-088504-9.00100-8
Nicaud J-M (2012) Yarrowia lipolytica. Yeast 29:409–418. https://doi.org/10.1002/yea.2921
Nunes PMB, da Rocha SM, Amaral PFF, da Rocha-Leão MHM (2013) Study of trans–trans farnesol effect on hyphae formation by Yarrowia lipolytica. Bioprocess Biosyst Eng 36:1967–1975. https://doi.org/10.1007/s00449-013-0973-8
Papagianni M, Joshi N, Moo-Young M (2002) Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures. J Ind Microbiol Biotechnol 29:259–263. https://doi.org/10.1038/sj.jim.7000289
Park C-H, Okos MR, Wankat PC (1989) Acetone–butanol–ethanol (ABE) fermentation in an immobilized cell trickle bed reactor. Biotechnol Bioeng 34:18–29. https://doi.org/10.1002/bit.260340104
Pignède G, Wang H, Fudalej F, Gaillardin C, Seman M, Nicaud J-M (2000) Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica. J Bacteriol 182:2802–2810
Querol A, Barrio E, Huerta T, Ramón D (1992) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol 58:2948–2953
Ramos-Sánchez LB, Cujilema-Quitio MC, Julian-Ricardo MC, Cordova J, Fickers P (2015) Fungal lipase production by solid-state fermentation. J Bioprocess Biotech 5:203. https://doi.org/10.4172/2155-9821.1000203
Rodriguez C, Domínguez A (1984) The growth characteristics of Saccharomycopsis lipolytica: morphology and induction of mycelium formation. Can J Microbiol 30:605–612. https://doi.org/10.1139/m84-090
Rosche B, Li XZ, Hauer B, Schmid A, Buehler K (2009) Microbial biofilms: a concept for industrial catalysis? Trends Biotechnol 27:636–643. https://doi.org/10.1016/j.tibtech.2009.08.001
Ruiz-Herrera J, Sentandreu R (2002) Different effectors of dimorphism in Yarrowia lipolytica. Arch Microbiol 178:477–483. https://doi.org/10.1007/s00203-002-0478-3
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, NY, USA
Sassi H, Delvigne F, Kar T, Nicaud J-M, Crutz-Le Coq A-M, Steels S, Fickers P (2016) Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica. Microb Cell Factories 15. https://doi.org/10.1186/s12934-016-0558-8
Sudbery P, Gow N, Berman J (2004) The distinct morphogenic states of Candida albicans. Trends Microbiol 12:317–324. https://doi.org/10.1016/j.tim.2004.05.008
Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9. https://doi.org/10.1016/j.ymben.2012.08.007
Timoumi A, 2017. Study of the dynamics of physiological and metabolic responses of Yarrowia lipolytica to environmental physico-chemical perturbations. PhD thesis, INSA, Toulouse, France. https://hal.inria.fr/tel-01628212v1
Todhanakasem T, Sangsutthiseree A, Areerat K, Young GM, Thanonkeo P (2014) Biofilm production by Zymomonas mobilis enhances ethanol production and tolerance to toxic inhibitors from rice bran hydrolysate. New Biotechnol 31:451–459. https://doi.org/10.1016/j.nbt.2014.06.002
Urbance SE, Pometto AL, DiSpirito AA, Denli Y (2004) Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using plastic composite support bioreactors. Appl Microbiol Biotechnol 65:664–670. https://doi.org/10.1007/s00253-004-1634-2
Vandermies M, Denies O, Nicaud J-M, Fickers P (2017) EYK1 encoding erythrulose kinase as a catabolic selectable marker for genome editing in the non-conventional yeast Yarrowia lipolytica. J Microbiol Methods 139:161–164. https://doi.org/10.1016/j.mimet.2017.05.012
Wightman R, Bates S, Amornrrattanapan P, Sudbery P (2004) In Candida albicans, the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization. J Cell Biol 164:581–591. https://doi.org/10.1083/jcb.200307176
Willaert R (2011) Cell immobilization and its applications in biotechnology. In: Allman A (ed) Fermentation Microbiology and Biotechnology, 3rd edn. CRC Press, Boca Raton, USA, pp 313–367. https://doi.org/10.1201/b11490-13
Zune Q, Delepierre A, Gofflot S, Bauwens J, Twizere J-C, Punt PJ, Francis F, Toye D, Bawin T, Delvigne F (2015) A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae. Appl Microbiol Biotechnol 99:6241–6254. https://doi.org/10.1007/s00253-015-6608-z
Zune Q, Soyeurt D, Toye D, Ongena M, Thonart P, Delvigne F (2014) High-energy X-ray tomography analysis of a metal packing biofilm reactor for the production of lipopeptides by Bacillus subtilis. J Chem Technol Biotechnol 89:382–390. https://doi.org/10.1002/jctb.4128
Acknowledgements
M. Vandermies and F. Carly are recipients of a fellowship from the Fonds pour la Formation à la Recherche dans l’Industrie et l’Agriculture (FRIA).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Vandermies, M., Kar, T., Carly, F. et al. Yarrowia lipolytica morphological mutant enables lasting in situ immobilization in bioreactor. Appl Microbiol Biotechnol 102, 5473–5482 (2018). https://doi.org/10.1007/s00253-018-9006-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-9006-5