Genetic modification of enzymes and metabolic pathways for the improvement of fatty acid synthesis in the yeast Saccharomyces cerevisiae Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biowissenschaften der Goethe-Universität in Frankfurt am Main von Renata Pavlović aus Kakanj, Bosnien-Herzegowina Frankfurt am Main 2016 (D 30) vom Fachbereich Biowissenschaften der Goethe-Universität Frankfurt als Dissertation angenommen. Dekanin: Prof. Dr. Meike Piepenbring Gutachter: Prof. Dr. Eckhard Boles Prof. Dr. Martin Grininger Datum der Disputation: 28. Februar 2017 The mystery of human existence lies not in just staying alive, but in finding something to live for. Fyodor Dostoyevsky Table of contents Summary __________________________________________________________________ 1 1. Introduction ___________________________________________________________ 3 Fatty acid biosynthesis and degradation ________________________________________ 4 1.1.2 Fatty acid biosynthesis in S. cerevisiae ______________________________________________________ 4 1.1.3 Elongation and synthesis of unsaturated fatty acids ___________________________________________ 6 1.1.4 Fatty acids as energy storage ______________________________________________________________ 8 1.1.5 Degradation and export of fatty acids _______________________________________________________ 9 Metabolic engineering ______________________________________________________ 10 1.2.1 Fermentation medium __________________________________________________________________ 10 1.2.2 Oleaginous microorganisms ______________________________________________________________ 10 1.2.3 Pathway optimization___________________________________________________________________ 12 Aim of this work ___________________________________________________________ 16 2. Materials and Methods _________________________________________________ 17 Medium, microorganisms and cultivation ______________________________________ 17 2.1.1 Medium ______________________________________________________________________________ 17 2.1.2 Microorganisms and their cultivation ______________________________________________________ 18 Plasmids _________________________________________________________________ 19 Oligonucleotides __________________________________________________________ 24 Materials, chemicals and equipment __________________________________________ 24 Molecular biological methods ________________________________________________ 26 2.5.1 Isolation of plasmid DNA from E. coli ______________________________________________________ 26 2.5.2 Isolation of plasmid and genomic DNA from S. cerevisiae ______________________________________ 26 2.5.3 Determination of DNA concentration ______________________________________________________ 27 2.5.4 Digestion of DNA with restriction endonucleases (restriction digestion) __________________________ 27 2.5.5 Polymerase chain reaction (PCR) __________________________________________________________ 28 2.5.6 Agarose gel-electrophoresis for DNA separation _____________________________________________ 28 2.5.7 DNA-purification and DNA-extraction form agarose gels _______________________________________ 28 2.5.8 DNA sequencing _______________________________________________________________________ 28 Genetic methods __________________________________________________________ 29 2.6.1 Transformation of E. coli ________________________________________________________________ 29 2.6.2 Transformation of S. cerevisiae ___________________________________________________________ 29 2.6.3 Codon optimization of genes _____________________________________________________________ 30 2.6.4 Plasmid construction by recombinational cloning ____________________________________________ 30 2.6.5 Plasmid construction by Gibson assembly __________________________________________________ 30 I Table of contents 2.6.6 Genomic gene deletion by Delitto Perfetto__________________________________________________ 31 2.6.7 Genomic gene deletion by CRISPR/Cas9 ____________________________________________________ 31 Methods for cell cultivation and fermentation experiments ________________________ 32 2.7.1 Spectrophotometrical determination of cell density __________________________________________ 32 2.7.2 Glycerol stock cultures __________________________________________________________________ 32 2.7.3 Serial dilution spot assay ________________________________________________________________ 33 2.7.4 Aerobic batch fermentations _____________________________________________________________ 33 2.7.5 Aerobic batch fermentations with ion exchanger _____________________________________________ 33 2.7.6 Lyophilisation of cell pellets and determination of cell dry weight _______________________________ 33 Metabolite analysis by HPLC _________________________________________________ 34 Fatty acid analysis _________________________________________________________ 34 2.9.1 Extraction of lipids from culture medium ___________________________________________________ 34 2.9.2 Extraction of lipids from dry cell pellet _____________________________________________________ 34 2.9.3 Preparation of fatty acid methyl esters _____________________________________________________ 35 2.9.4 Determination of fatty acid methyl esters by GC _____________________________________________ 35 2.9.5 Preparation of fatty acids with DMDS ______________________________________________________ 35 2.9.6 Fatty acid analysis by GC-MS _____________________________________________________________ 36 2.9.7 Thin layer chromatography (TLC) __________________________________________________________ 36 In Silico Methods & Software ________________________________________________ 36 3. Results _______________________________________________________________ 37 Desaturases ______________________________________________________________ 37 3.1.1 Screening for desaturases with terminal desaturase activity ____________________________________ 37 3.1.2 Site directed mutagenesis of Δ9 desaturase Ole1p ___________________________________________ 44 Improving fatty acid synthesis _______________________________________________ 48 3.2.1 Optimization of acetyl-CoA synthesis ______________________________________________________ 48 3.2.2 Increasing fatty acid content by DGA1 and TGL3 expression ____________________________________ 50 Medium chain fatty acid synthesis ____________________________________________ 53 3.3.1 Effect of FAS mutations on fatty acid synthesis ______________________________________________ 53 3.3.2 Effect of culture medium on fatty acid synthesis _____________________________________________ 60 3.3.3 Effect of promoter exchange on fatty acid synthesis __________________________________________ 62 3.3.4 Optimization of MCFA synthesis by metabolic engineering _____________________________________ 67 3.3.4.1 Deletion of acyl-CoA synthetase Faa2p ___________________________________________________ 67 3.3.4.2. Degradation of triacylglycerol __________________________________________________________ 69 3.3.5 Extraction of MCFA from medium with ion exchanger _________________________________________ 72 3.3.6. Analysis of putative hydrolases for MCFA production _________________________________________ 76 3.3.6.1 Identification of putative hydrolases _____________________________________________________ 76 3.3.6.2 Effect of EEB1, EHT1 and MGL2 deletion __________________________________________________ 77 II Table of contents 4. Discussion ____________________________________________________________ 82 Desaturases ______________________________________________________________ 82 4.1.1 Characterization of fungi desaturases ______________________________________________________ 83 4.1.2 Site directed mutagenesis in Ole1p ________________________________________________________ 85 Improving fatty acid synthesis in general _______________________________________ 86 4.2.1 Implementation of co-substrate and metabolite synthesis _____________________________________ 86 4.2.2 Deletion of fatty acyl-CoA synthetases _____________________________________________________ 88 Medium chain fatty acids ___________________________________________________ 89 4.3.1 Selective production of MCFA by reprograming of the FAS _____________________________________ 89 4.3.2 Culture medium composition ____________________________________________________________ 93 4.3.3 Change expression pattern of mutated FAS _________________________________________________ 95 4.3.4 Best approach for optimal yield of MCFA ___________________________________________________ 97 4.3.5 Improved metabolic fluxes towards MCFA synthesis __________________________________________ 98 4.3.6 Three acyl-CoA thioesterase with different substrate spectrum ________________________________ 100 4.3.7 Continuous product removal with a ion exchange resins ______________________________________ 102 Outlook _________________________________________________________________ 102 5. Zusammenfassung ____________________________________________________ 104 6. References ___________________________________________________________ 109 7. Appendices __________________________________________________________ 128 I. Abbreviations ____________________________________________________________ 128 II. DNA oligonucleotides ______________________________________________________ 131 III. Supplemental figures and tables _____________________________________________ 140 IV. Acknowledgements _______________________________________________________