Carlsen, Simon

Eliasson Lantz, Anna

Kielland-Brandt, Morten

Nielsen, Michael Lynge

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 17, 2017Establishing Saccharomyces cerevisiae as platform for isoprenoid productionCarlsen, Simon; Nielsen, Michael Lynge; Kielland-Brandt, Morten; Eliasson Lantz, AnnaPublication date:2009Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Carlsen, S., Nielsen, M. L., Kielland-Brandt, M., & Eliasson Lantz, A. (2009). Establishing Saccharomycescerevisiae as platform for isoprenoid production. Poster session presented at Symposium for BiotechnologicalResearch, Lyngby, .Directed Evolution towards Increased IsoprenoidProduction in Saccharomyces cerevisiaeSimon Carlsen, Morten C. Kielland-Brandt, Michael Lynge Nielsen and Anna Eliasson LantzCenter for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark E-mail: SimC@bio.dtu.dkIntroductionOrganic synthesis of isoprenoids often results in low yields due to their complex structure. This makes production in large scale, by organicsynthesis economically unfeasible. Microbial production can easily be scaled to meet current demands and it is also an environmental benignproduction method compared to organic synthesis. Thus it would be attractive to engineer a microorganism to produce high amounts of IPPand other immediate prenyl precursors such as geranyl pyrophosphate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, therebyestablishing it as a microbial platform for heterologous expression of isoprenoid genes (see figure 1).This project is focused on creating diversity within a lycopene producing S. cerevisiae strain by construction of gDNA-, cDNA-, and transposon-libraries. The diversified population of S. cerevisiae clones will afterwards be screened using the isoprenoid molecule lycopene as a modelcompound, hereby enabling the isolation of phenotypes producing higher amounts of isoprenoid. This will elucidate novel genetic targets forincreasing isoprenoid production in S. cerevisiae.Future perspectivesWhen different genetic perturbations that increase the production of isoprenoids have been identified, the underlying metabolic mechanismswill be sought identified through further analysis. We foresee that some perturbations will be straight forward to identify e.g. overexpression ofgenes which are directly linked to isoprenoid precursor biosynthesis. The mechanisms which are harder to unravel will be the ones encoded byORFs with unassigned functions or regulatory mechanisms. Finally systematic combinations of the identified perturbations will be engineeredaiming at the construction of a S. cerevisiae platform for high titer production of isoprenoids.Figure 2: Method for construction of gDNA library. In order to construct gDNA libraries it is essential to obtain randomly digested gDNA. This can be done bytreating high concentration gDNA with a 4 bp recognition endonuclease for a defined period of time. To find the correct amount of enzyme needed to obtain a partialdigest a dilution series is setup and the reactions with the desired size range is subsequently pooled wherefrom the size selected gDNA pieces are purified. Therandom pieces are then ligated into an expression cassette (TEF1p-gDNA-CYC1t) in a single copy vector (pRS414TEF from Mumberg et al. 1994) followed bytransformation of ultra competent Escherichia coli cells. The final gDNA library is purified from the E. coli transformants. This library can be used for transformationof Saccharomyces cerevisiae. This method is based on Jauert et al. 2005 and Liu 2002.gDNA library constructionpRS414TEF5 41 4 bpTRP1 Te rTRP1ApRTRP1 ProP TEF1CEN6ARSH4F1 oriPM B1T CY C1BamHI (3 208)SpeI (3 202)Xho I (3 25 9)10 kb5 kb0.5 kb1 kbSau3AI partial digest of gDNARandom pieces of gDNAClone into single copy expression vectorTransform ultra competent E. coligDNA libraryReferencesJauert, P. A.; Jensen, L. E. and Kirkpatrick, D. T. A novel yeast genomic DNA library on a geneticin-resistance vector. Yeast. 2005, 22, 653-657.Liu, H. Constructing Yeast Libraries. Methods in Enzymology, vol 350. Part B. Edited by Guthrie, C. and Fink, G. R. Chapter 3. 2002, 72-86. ISBN: 0-12-182253-2.Mumberg, D.; Müller, R. and Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1994, 156, 119-122.Figure 3: A. Library screening based on lycopene colony coloration. The colonies exhibiting phenotypes with more intense red coloration compared to the wild typeare producing more lycopene than the wild type, hence the underlying genotype could result in a higher flux towards and through the mevalonate pathway herebyincreasing the precursor availability or in another way be beneficial for isoprenoid production. The transformants which shows intensified coloration can be furtheranalysed resulting in identification of the best candidates from which the underlying genotype can be elucidated. B. Biosynthetic pathway for converting farnesylpyrophosphate into lycopene. C. pSC203 plasmid for chromosomal integration of the CrtE-, CrtB-, and CrtI-gene from Erwinia herbicola under the control of theTEF1-, PGK1-, and GDP-promoter respectively. D. UV-Vis absorption spectrum for lycopene. E. S. cerevisiae transformed with the pSC203 plasmid.gDNA librariesTransposon libraries cDNA librariesLycopeneOPPGGPPOPPFPPCRTE (GGPP synthetase)CRTB (Phytoene synthetase)PhytoeneCRTI (Phytoene desaturase)pSC20310573 bpcrtBcrtECRTITerURA3APrPGK1pP TEF1PromoterP GPDF1 oriCOLE1ACT1tADH1tT CYC0255075100225 325 425 525%Wavelength (nm)A BCDETransformationColony screeningMicro titer screeningShake flask characterizationTest fermentationDataClonesNatureS. cerevisiae cell factoryTransfer genes of interestCarbon sourceFermentationPharmaceutical or precursorsFigure 1: Setup of S. cerevisiae as microbial production platform to produce secondary metabolites. By transferring the genes encoding the isoprenoids of interestfrom its native host to a S. cerevisiae strain expressing them heterologous, it becomes possible to construct a microbial production platform for production ofisoprenoids, which could be used as pharmaceuticals or precursors instead of having to rely on extraction from native sources (thin curved arrow). By fermentationof the constructed strain it will be possible to convert cheap carbon sources such as glucose into valuable products.Yeast as cell factory - concept

Establishing Saccharomyces cerevisiae as platform for isoprenoid production

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Establishing Saccharomyces cerevisiae as platform for isoprenoid production

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