2,3-Butanediol production using acetogenic bacteria Dissertation Submitted for the fulfillment of the requirements for the doctoral degree Dr. rer. nat. at the Faculty of Natural Sciences, Ulm University Catarina Erz from Stuttgart – Bad Cannstatt 2017 The present study was performed at the Institute of Microbiology and Biotechnology, Ulm University, under the direction of Prof. Dr. Peter Dürre. Faculty dean in office: Prof. Dr. Peter Dürre First reviewer: Prof. Dr. Peter Dürre Second reviewer: Prof. Dr. Bernhard J. Eikmanns Date of the doctoral examination: October 11 th , 2017 Content Content Abbreviations ____________________________________________________ I 1. Introduction ___________________________________________________ 1 2. Material and Methods __________________________________________ 10 2.1 Microorganisms, plasmids, and primers ____________________________10 2.1.1 Bacterial strains _________________________________________________ 10 2.1.2 Vectors and plasmids ____________________________________________ 11 2.1.3 Primers ________________________________________________________ 14 2.2 Chemicals, enzymes, and gases ___________________________________17 2.3 Bacterial cultivation ____________________________________________19 2.3.1 Cultivation media _______________________________________________ 19 2.3.1.1 Modified 1.0 ATCC 1612 medium ________________________________ 19 2.3.1.2 Modified ATCC 1612 medium ___________________________________ 22 2.3.1.3 Lyophilization medium ________________________________________ 22 2.3.1.4 Lysogeny broth medium -modified- ______________________________ 23 2.3.1.5 Rajagopalan medium -modified- _________________________________ 23 2.3.1.6 Super optimal broth medium -modified- __________________________ 24 2.3.1.7 Tanner medium -modified- _____________________________________ 25 2.3.1.8 Yeast tryptone fructose medium -modified-________________________ 25 2.3.2 Antibiotic selection ______________________________________________ 26 2.4 Growth conditions _____________________________________________27 2.4.1 Aerobic cultivation ______________________________________________ 27 2.4.2 Anaerobic cultivation ____________________________________________ 27 2.4.3 Strain conservation ______________________________________________ 28 Content 2.4.3.1 Glycerol ____________________________________________________ 28 2.4.3.2 SMP buffer/DMSO ___________________________________________ 28 2.4.3.3 Lyophilization _______________________________________________ 29 2.4.4 Determination of growth and metabolic production parameters _________ 29 2.4.4.1 Determination of optical density ________________________________ 29 2.4.4.2 Quantification of substrate and microbial metabolic products by high performance liquid chromatography ____________________________ 30 2.4.4.3 Quantification of microbial metabolic products by gas chromatography _ 31 2.5 Nucleic acid methods___________________________________________ 32 2.5.1 Nucleic acid isolation ____________________________________________ 32 2.5.1.1 Plasmid isolation from E. coli using the Zyppy TM Plasmid Miniprep Kit ___ 32 2.5.1.2 Plasmid isolation from E. coli using QIAGEN Plasmid Midi Kit __________ 32 2.5.1.3 Genomic DNA isolation from Gram positive bacteria using MasterPure TM Gram Positive DNA Purification Kit ______________________________ 33 2.5.1.4 RNA isolation from clostridia ___________________________________ 33 2.5.2 Polymerase chain reaction ________________________________________ 35 2.5.2.1 Composition of PCR reaction mixtures with different DNA polymerases _ 35 2.5.2.2 Standard PCR program ________________________________________ 36 2.5.2.3 Insertion of restriction recognition sequences via PCR _______________ 37 2.5.3 DNA modification _______________________________________________ 37 2.5.3.1 Restriction enzyme digestion of DNA _____________________________ 37 2.5.3.2 Dephosphorylation of digested plasmids __________________________ 38 2.5.3.3 DNA ligation ________________________________________________ 38 2.5.3.4 In-Fusion® HD Cloning Kit ______________________________________ 39 2.5.3.5 Radioactive labelling of DNA fragments ___________________________ 39 Content 2.5.4 Purification of nucleic acids _______________________________________ 40 2.5.4.1 Purification of amplified and digested DNA ________________________ 40 2.5.4.2 Purification of radioactively labelled DNA _________________________ 41 2.5.4.3 Purification of ligated DNA by microdialysis ________________________ 41 2.5.5 Gel electrophoresis ______________________________________________ 41 2.5.5.1 Non-denaturating gel electrophoresis ____________________________ 41 2.5.5.2 Denaturating gel electrophoresis ________________________________ 42 2.5.6 Determination of nucleic acid concentration __________________________ 43 2.6 DNA sequencing and data analysis ________________________________43 2.6.1 Sequencing of vectors and DNA fragments ___________________________ 43 2.6.2 Genome sequencing _____________________________________________ 44 2.6.3 Mapping for single nucleotide polymorphism (SNP) analysis _____________ 44 2.6.4 Comparison of locus tags of different acetogens _______________________ 44 2.7 DNA transfer in bacteria _________________________________________45 2.7.1 DNA transfer in Escherichia coli ____________________________________ 45 2.7.1.1 Preparation of electrocompetent E. coli cells _______________________ 45 2.7.1.2 Preparation of fast competent E. coli cells _________________________ 45 2.7.1.3 Transformation of electrocompetent E. coli cells ____________________ 45 2.7.1.4 Preparation of chemically competent E. coli cells ___________________ 46 2.7.1.5 Transformation of chemically competent E. coli cells ________________ 47 2.7.2 DNA transfer in A. woodii and Clostridium by electroporation____________ 47 2.7.2.1 Preparation of electrocompetent A. woodii and Clostridium cells _______ 47 2.7.2.2 Transformation of electrocompetent A. woodii and Clostridium ________ 47 2.7.3 DNA transfer in Clostridium by conjugation ___________________________ 48 2.7.4 Northern blot ___________________________________________________ 49 Content 2.7.4.1 Transfer of RNA onto a nylon membrane __________________________ 49 2.7.4.2 Hybridization of radioactively labelled probe with RNA ______________ 50 3. Results ______________________________________________________ 53 3.1 Improvement of 2,3-BD production in acetogenic bacteria ____________ 53 3.1.1 Natural 2,3-BD production in acetogenic bacteria _____________________ 53 3.1.2 Construction of plasmids for enhancement of natural 2,3-BD production __ 54 3.1.3 Homologous 2,3-BD overproduction in C. ljungdahlii ___________________ 56 3.1.4 Homologous 2,3-BD overproduction in C. coskatii _____________________ 60 3.1.5 Modifications of 2,3-BD production plasmid pJIR750_23BD _____________ 63 3.1.5.1 Removal of a potential terminator structure _______________________ 64 3.1.5.2 Exchange of bdh with adh (primary-secondary alcohol dehydrogenase) _ 66 3.1.5.3 BudABC operon from Raoultella terrigena _________________________ 68 3.1.5.4 Growth experiments of different C. ljungdahlii strains harboring the modified 2,3-BD plasmids ______________________________________________ 70 3.1.6 Sequencing of C. ljungdahlii [pMTL82151] and C. ljungdahlii WT with SNP analysis _______________________________________________________ 75 3.1.7 Detection and quantification of transcripts responsible for 2,3-BD production __________________________________________________________________ 77 3.1.8 Heterologous 2,3-BD production in A. woodii _________________________ 79 3.1.9 Enhancement of 2,3-BD production by overexpressing nifJ ______________ 82 3.2 Heterologous butanol production ________________________________ 90 4. Discussion ____________________________________________________ 95 4.1 Natural microbial 2,3-BD production ______________________________ 95 4.2 Enhancement of 2,3-BD production using acetogens ________________ 103 Content 4.2.1 Enhancement of 2,3-BD production via overexpression of alsS , budA , and bdh __________________________________________________________________ 103 4.2.2 Modifications of 2,3-BD production plasmid to optimize 2,3-BD production 107 4.2.3 PFOR enzyme as potential bottle-neck in 2,3-BD production? ___________ 112 4.3 Detection of transcripts from synthetic 2,3-BD operons in C. ljungdahlii 116 4.4 Investigation of mutated C. ljungdahlii [pMTL82151] showing increased flux towards ethanol and 2,3-BD ___________________________________ 118 4.5 A. woodii as suitable host for 2,3-BD production from H 2 + CO 2? ______ 122 4.6 Heterologous butanol production in C. ljungdahlii - the conflict of large plasmid transformation _______________________________________ 124 5. Summary ___________________________________________________ 127 6. Zusammenfassung ____________________________________________ 129 7. References __________________________________________________ 132 8. Supplement _________________________________________________ 159 Contribution to this work ________________________________________ 165 Danksagung ___________________________________________________ 167 Curriculum vitae ________________________________________________ 171 Statement_____________________________________________________ 173 Abbreviations I Abbreviations A adenosine A. Acetobacterium ; Aeromonas ; Aspergillus AA amino acid abbr. abbreviation ad . addiere (fill up to) ADP adenosine diphosphate AG Aktiengesellschaft (joint-stock company) AG & Co. Aktiengesellschaft