Cyanobacteria as biocatalysts for the production of volatile hydrocarbons

_____________________________________________________ 8 TIIVISTELMÄ _____________________________________________________ 9 1.Introduction __________________________________________________ 11 1.1 Towards sustainable production of carbon-based chemicals _________ 11 1.1.1 The need to move from a linear to circular economy ______________________ 11 1.1.2 Biomass-based systems to produce hydrocarbon substitutes _______________ 12 1.1.3 Cyanobacteria as photosynthetic cell factories for the sustainable production of valuable chemicals _________________________________________________ 16 1.2 Ethylene production in vivo ___________________________________ 18 1.2. Ethylene biosynthesis in plants and microbes ____________________________ 18 1.2. 2 The ethylene-forming enzyme (EFE) and 2-oxoglutarate-dependent pathway _ 19 1.2. 3 Heterologous expression of the ethylene-forming enzyme gene in cyanobacteria and genetic instability _______________________________________________ 20 1.3 Cyanobacteria and aldehyde deformylating oxygenase _____________ 24 1.3.1 Aldehyde deformylating oxygenase ____________________________________ 25 1.3.2 Synthetic pathways for short alkane production using aldehyde deformylating oxygenase ________________________________________________________ 26 1.3.3 Aldehyde deformylating oxygenase as the main bottleneck to propane production_______________________________________________________ 29 2.Aims ________________________________________________________ 33 3.Materials and methods _________________________________________ 34 3.1 Expression vectors and strains _________________________________ 34 3.2 Culture conditions ___________________________________________ 34 3.3 GC-MS quantitation of volatile hydrocarbons and alcohols in vivo ____ 35 3.4 RNA isolation and cDNA synthesis ______________________________ 35 3.5 Real-Time quantitative PCR ___________________________________ 36 3.6 Protein quantification ________________________________________ 36 3.7 Spectrophotometric analysis __________________________________ 36 3.8 Ethylene toxicity test ________________________________________ 36 4.Overview of results ____________________________________________ 37 4.1 Ethylene-forming enzyme _____________________________________ 37 4.1.1 Ethylene-forming enzyme as a reporter protein for promoter screening ______ 37 4.1.2 Codon optimization of the ethylene-forming enzyme gene is not a pre-requisite for the stable production of ethylene in Synechocystis sp. PCC 6803 _________ 39 4.1.3 Strategy for the stable production of ethylene in Synechococcus sp. 7942 _____ 40 4.2 In vivo comparison of five cyanobacterial aldehyde deformylating oxygenase homologs in E. coli for the production of short-chain alkanes __________________________________________________________ 41 5.Discussion ____________________________________________________ 43 5.1 Use of ethylene-forming enzyme as a quantitative reporter in cyanobacteria ______________________________________________ 43 5.2 Genetic instability of ethylene-forming enzyme gene in cyanobacteria_ 45 5.2. 1 Codon optimization vs. original sequence ________________________ 45 5.2. 2 The role of the PsbA gene family and its relationship with the reported genetic instability of the ethylene-forming enzyme gene in Synechococcus __________ 48 5.3 Aldehyde deformylating oxygenase and the microbial production of volatile alkanes _____________________________________________ 50 6.Concluding remarks ____________________________________________ 52 7.Acknowledgements ____________________________________________ 53 8.References ___________________________________________________ 54 31042434_Thesis_Veronica_Carbonell_inside_19_03_01_B. indd 4 1.3.2019 16:26:07