Skip to main content
SpringerLink
Log in

Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola

  • Protocol
  • First Online:
Synthetic Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1772))

Abstract

In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Solaiman DKY, Ashby RD, Crocker NV (2015) High-titer production and strong antimicrobial activity of sophorolipids from Rhodotorula bogoriensis. Biotechnol Prog 31(4):867–874

    Article  CAS  PubMed  Google Scholar 

  2. Ashby RD, Solaiman DKY, Foglia TA (2008) Property control of sophorolipids: influence of fatty acid substrate and blending. Biotechnol Lett 30(6):1093–1100

    Article  CAS  PubMed  Google Scholar 

  3. Van Bogaert I, Fleurackers S, Van Kerrebroeck S, Develter D, Soetaert W (2011) Production of new-to-nature sophorolipids by cultivating the yeast Candida bombicola on unconventional hydrophobic substrates. Biotechnol Bioeng 108(4):734–741

    Article  PubMed  Google Scholar 

  4. Zhang J, Saerens KMJ, Van Bogaert INA, Soetaert W (2011) Vegetable oil enhances sophorolipid production by Rhodotorula bogoriensis. Biotechnol Lett 33(12):2417–2423

    Article  CAS  PubMed  Google Scholar 

  5. Van Bogaert INA, De Maeseneire SL, Develter D, Soetaert W, Vandamme EJ (2008) Development of a transformation and selection system for the glycolipid-producing yeast Candida bombicola. Yeast 25(4):273–278

    Article  PubMed  Google Scholar 

  6. Saerens KMJ, Zhang J, Saey L, Van Bogaert INA, Soetaert W (2011) Cloning and functional characterization of the UDP-glucosyltransferase UgtB1 involved in sophorolipid production by Candida bombicola and creation of a glucolipid-producing yeast strain. Yeast 28(4):279–292

    Article  CAS  PubMed  Google Scholar 

  7. Van Bogaert INA, Buyst D, Martins JC, Roelants SLKW, Soetaert WK (2016) Synthesis of bolaform biosurfactants by an engineered Starmerella bombicola yeast. Biotechnol Bioeng 113(12):2644–2651

    Article  PubMed  Google Scholar 

  8. Saerens KMJ, Saey L, Soetaert W (2011) One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola. Biotechnol Bioeng 108(12):2923–2931

    Article  CAS  PubMed  Google Scholar 

  9. Roelants SLKW, Saerens KMJ, Derycke T, Li B, Lin Y-C, Van de Peer Y, De Maeseneire SL, Van Bogaert INA, Soetaert W (2013) Candida bombicola as a platform organism for the production of tailor-made biomolecules. Biotechnol Bioeng 110(9):2494–2503

    Article  CAS  PubMed  Google Scholar 

  10. Van Bogaert INA, Holvoet K, Roelants SLKW, Li B, Lin Y-C, Van de Peer Y, Soetaert W (2013) The biosynthetic gene cluster for sophorolipids: a biotechnological interesting biosurfactant produced by Starmerella bombicola. Mol Microbiol 88(3):501–509

    Article  PubMed  Google Scholar 

  11. Saerens KMJ, Roelants SLKW, Van Bogaert INA, Soetaert W (2011) Identification of the UDP-glucosyltransferase gene UGTA1, responsible for the first glucosylation step in the sophorolipid biosynthetic pathway of Candida bombicola ATCC 22214. FEMS Yeast Res 11(1):123–132

    Article  CAS  PubMed  Google Scholar 

  12. Roelants SLKW, Ciesielska K, De Maeseneire SL, Moens H, Everaert B, Verweire S, Denon Q, Vanlerberghe B, Van Bogaert INA, Van der Meeren P, Devreese B, Soetaert W (2016) Towards the industrialization of new biosurfactants: biotechnological opportunities for the lactone esterase gene from Starmerella bombicola. Biotechnol Bioeng 113(3):550–559

    Article  CAS  PubMed  Google Scholar 

  13. Ciesielska K, Van Bogaert IN, Chevineau S, Li B, Groeneboer S, Soetaert W, Van de Peer Y, Devreese B (2014) Exoproteome analysis of Starmerella bombicola results in the discovery of an esterase required for lactonization of sophorolipids. J Proteome 98:159–174

    Article  CAS  Google Scholar 

  14. Van Bogaert INA, De Maeseneire SL, De Schamphelaire W, Develter D, Soetaert W, Vandamme EJ (2007) Cloning, characterization and functionality of the orotidine-5′-phosphate decarboxylase gene (URA3) of the glycolipid-producing yeast Candida bombicola. Yeast 24(3):201–208

    Article  PubMed  Google Scholar 

  15. Friel D, Vandenbol M, Haïssam Jijakli M (2003) Cloning and sequence analysis of the TRP1 gene encoding the phosphoribosyl anthranilate isomerase from Pichia anomala (strain K). Yeast 20(16):1331–1337

    Article  CAS  PubMed  Google Scholar 

  16. Pronk JT (2002) Auxotrophic yeast strains in fundamental and applied research. Appl Environ Microbiol 68(5):2095–2100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Rodríguez L, Chávez FP, González ME, Basabe L, Rivero T (1998) Isolation and sequence analysis of the orotidine-5′-phosphate decarboxylase gene (URA3) ofCandida utilis. Comparison with the OMP decarboxylase gene family. Yeast 14(15):1399–1406

    Article  PubMed  Google Scholar 

  18. François F, Chapeland-Leclerc F, Villard J, Noël T (2004) Development of an integrative transformation system for the opportunistic pathogenic yeastCandida lusitaniae usingURA3 as a selection marker. Yeast 21(2):95–106

    Article  PubMed  Google Scholar 

  19. De Maeseneire SL, De Groeve MRM, Dauvrin T, De Mey M, Soetaert W, Vandamme EJMC (2006) Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5′-monophosphate decarboxylase gene. FEMS Microbiol Lett 261(2):262–271

    Article  PubMed  Google Scholar 

  20. Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2(4):229–236

    Article  CAS  PubMed  Google Scholar 

  21. Basehoar AD, Zanton SJ, Pugh BF (2004) Identification and distinct regulation of yeast TATA box-containing genes. Cell 116(5):699–709

    Article  CAS  PubMed  Google Scholar 

  22. GUO Z, SHERMAN F (1996) 3′-end-forming signals of yeast mRNA. Trends Biochem Sci 21(12):477–481

    Article  CAS  PubMed  Google Scholar 

  23. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):31–34

    Article  CAS  PubMed  Google Scholar 

  24. Ruiz-Diez B (2002) Strategies for the transformation of filamentous fungi. J Appl Microbiol 92(2):189–195

    Article  CAS  PubMed  Google Scholar 

  25. Van Bogaert INA (2008) Microbial synthesis of sophorolipids by the yeast Candida bombicola, Ghent University

    Google Scholar 

  26. Hentges P, Van Driessche B, Tafforeau L, Vandenhaute J, Carr AM (2005) Three novel antibiotic marker cassettes for gene disruption and marker switching inSchizosaccharomyces pombe. Yeast 22(13):1013–1019

    Article  CAS  PubMed  Google Scholar 

  27. Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21(14):3329–3330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Manivasakam P, Weber SC, McElver J, Schiestl RH (1995) Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res 23(14):2799–2800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Verbeke J, Beopoulos A, Nicaud J-M (2013) Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains. Biotechnol Lett 35(4):571–576

    Article  CAS  PubMed  Google Scholar 

  30. Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4(7):e6441

    Article  PubMed  PubMed Central  Google Scholar 

  31. Ianiri G, Wright SAI, Castoria R, Idnurm A (2011) Development of resources for the analysis of gene function in Pucciniomycotina red yeasts. Fungal Genet Biol 48(7):685–695

    Article  CAS  PubMed  Google Scholar 

  32. Liu Y, Koh CMJ, Sun L, Hlaing MM, Du M, Peng N, Ji L (2013) Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides. Appl Microbiol Biotechnol 97(2):719–729

    Article  CAS  PubMed  Google Scholar 

  33. Ianiri G, Idnurm A (2015) Agrobacterium tumefaciens- mediated transformation of Pucciniomycotina red yeasts. Springer, Cham, pp 163–168

    Google Scholar 

  34. Abbott EP, Ianiri G, Castoria R, Idnurm A (2013) Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts. Appl Microbiol Biotechnol 97(1):283–295

    Article  CAS  PubMed  Google Scholar 

  35. Kollár R, Reinhold BB, Petráková E, Yeh HJ, Ashwell G, Drgonová J, Kapteyn JC, Klis FM, Cabib E (1997) Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin. J Biol Chem 272(28):17762–17775

    Article  PubMed  Google Scholar 

  36. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197(2):345–346

    Article  CAS  PubMed  Google Scholar 

  37. Featherstone C, Jackson SP (1999) Ku, a DNA repair protein with multiple cellular functions? Mutat Res 434(1):3–15

    Article  CAS  PubMed  Google Scholar 

  38. Dynan WS, Yoo S (1998) Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 26(7):1551–1559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Näätsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS, Glieder A (2012) Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology. PLoS One 7(6):e39720

    Article  PubMed  PubMed Central  Google Scholar 

  40. Foureau E, Courdavault V, Rojas LF, Dutilleul C, Simkin AJ, Crèche J, Atehortùa L, Giglioli-Guivarc’h N, Clastre M, Papon N (2013) Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain. Biotechnol Lett 35(7):1035–1043

    Article  CAS  PubMed  Google Scholar 

  41. Saerens KMJ (2012) Synthesis of glycolipids by Candida bombicola, Ghent University

    Google Scholar 

  42. Roelants SLKW, Lodens S, Ciesielska K, Geys R, Coussement P, Pattyn F, Saerens K, Devreese B, De Maeseneire SL, Soetaert W (2018) Transformation of the biosurfactant production yeast Starmerella bombicola into a chassis organism: development, validation and application of a dedicated molecular toolkit.Metabolic Engineering (Submitted).

    Google Scholar 

  43. Yon J, Fried M (1989) Precise gene fusion by PCR. Nucleic Acids Res 17(12):4895

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yolov AA, Shabarova ZA (1990) Constructing DNA by polymerase recombination. Nucleic Acids Res 18(13):3983–3986

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zou R, Zhou K, Stephanopoulos G, Too HP (2013) Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA) method. PLoS One 8(11):e79557

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lang S, Brakemeier A, Heckmann R, Spöckner S, Rau U (2000) Production of native and modified sophorose lipids. Chim Oggi 18(10):76–79

    CAS  Google Scholar 

  47. Branduardi P, Valli M, Brambilla L, Sauer M, Alberghina L, Porro D (2004) The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications. FEMS Yeast Res 4(4–5):493–504

    Article  CAS  PubMed  Google Scholar 

  48. Roelants S (2013) Starmerella bombicola as a platform organism for the production of biobased compounds, Ghent University. Faculty of Bioscience Engineering

    Google Scholar 

  49. Geys R (2017) Engineering the metabolism of Starmerella bombicola for the production of tailor-made glycolipids, Ghent University

    Google Scholar 

  50. Hara A, Ueda M, Matsui T, Furuhashi K, Kanayama N, Tanaka A (1999) Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida Tropicalis. J Biosci Bioeng 87(6):717–720

    Article  CAS  PubMed  Google Scholar 

  51. Kanayama N, Ueda M, Atomi H, Tanaka A (1998) Genetic evaluation of physiological functions of thiolase isozymes in the n-alkane-assimilating yeast Candida tropicalis. J Bacteriol 180(3):690–698

    CAS  PubMed  PubMed Central  Google Scholar 

  52. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kuijpers NG, Solis-Escalante D, Bosman L, van den Broek M, Pronk JT, Daran J-M, Daran-Lapujade P (2013) A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences. Microb Cell Factories 12(1):47

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department of Biotechnology, Faculty of Bioscience Engineering,, Ghent University, Ghent, Belgium

    Sofie Lodens, Marilyn De Graeve, Sophie L. K. W. Roelants, Sofie L. De Maeseneire & Wim Soetaert

Authors
  1. Sofie Lodens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marilyn De Graeve
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sophie L. K. W. Roelants
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sofie L. De Maeseneire
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wim Soetaert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sophie L. K. W. Roelants .

Editor information

Editors and Affiliations

  1. Agilent Technologies, Inc, La Jolla, California, USA

    Jeffrey Carl Braman

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lodens, S., De Graeve, M., Roelants, S.L.K.W., De Maeseneire, S.L., Soetaert, W. (2018). Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola . In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7795-6_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7794-9

  • Online ISBN: 978-1-4939-7795-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation