Washington University in St. Louis Washington University Open Scholarship Engineering and Applied Science Theses & Dissertations McKelvey School of Engineering Winter 12-15-2019 Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass Drew Michael Delorenzo Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/eng_etds Part of the Biomedical Engineering and Bioengineering Commons, and the Microbiology Commons Recommended Citation Delorenzo, Drew Michael, "Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass" (2019). Engineering and Applied Science Theses & Dissertations. 492. https://openscholarship.wustl.edu/eng_etds/492 This Dissertation is brought to you for free and open access by the McKelvey School of Engineering at Washington University Open Scholarship. It has been accepted for inclusion in Engineering and Applied Science Theses & Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS McKelvey School of Engineering Department of Energy, Environmental, and Chemical Engineering Dissertation Examination Committee: Dr. Tae Seok Moon (chair) Dr. Gautam Dantas Dr. Marcus Foston Dr. Himadri Pakrasi Dr. Fuzhong Zhang Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass by Drew Michael DeLorenzo A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2019 St. Louis, Missouri © 2019, Drew Michael DeLorenzo Table of Contents Table of Figures........................................................................................................................... vii Table of Tables ............................................................................................................................. ix Acknowledgements ...................................................................................................................... xi Abstract of the Dissertation ...................................................................................................... xiii Chapter 1: Introduction ............................................................................................................... 1 1.1 Need for renewable sources of energy and chemicals ...................................................... 1 1.2 Sources of renewable energy and chemicals ..................................................................... 2 1.3 Lignocellulosic biomass as a sustainable carbon feedstock ............................................. 4 1.4 Taking advantage of lignin ................................................................................................. 6 1.5 Hybrid conversion of lignocellulose into value-added products ..................................... 9 1.6 Choosing Rhodococcus opacus PD630 as a biological catalyst ...................................... 10 1.7 Summary and organization of dissertation ..................................................................... 12 1.8 References .......................................................................................................................... 14 Chapter 2: Development of chemical and metabolite sensors for Rhodococcus opacus PD630 ........................................................................................................................................... 21 2.1 Abstract .............................................................................................................................. 21 2.2 Introduction ....................................................................................................................... 22 2.3 Results and discussion ....................................................................................................... 24 2.3.1 Fluorescent reporters ................................................................................................. 24 2.3.2 Chemically inducible promoters ............................................................................... 25 2.3.3 Nitrogen metabolite sensor ........................................................................................ 28 2.3.4 Aromatic metabolite sensors and insights into aromatic consumption pathways ............................................................................................................................... 30 2.4 Conclusion .......................................................................................................................... 35 2.5 Methods .............................................................................................................................. 35 2.5.1 Strains, plasmids and growth conditions .................................................................. 35 2.5.2 Fluorescence measurements ...................................................................................... 36 2.6 Supporting information .................................................................................................... 37 2.7 References .......................................................................................................................... 37 ii Chapter 3: Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630 .................................................................... 41 3.1 Abstract .............................................................................................................................. 41 3.2 Introduction ....................................................................................................................... 42 3.3 Results and discussion ....................................................................................................... 46 3.3.1 T7 RNAP expression platform. ................................................................................. 46 3.3.2 AND genetic logic gate ............................................................................................... 47 3.3.3 NAND genetic logic gate. ............................................................................................ 50 3.3.4 Synthetic IPTG inducible promoters. ....................................................................... 52 3.3.5 IMPLY genetic logic gate. .......................................................................................... 56 3.3.6 Aromatic sensors......................................................................................................... 57 3.4 Conclusion .......................................................................................................................... 60 3.5 Materials and methods ...................................................................................................... 61 3.5.1 Strains, plasmids and growth conditions .................................................................. 61 3.5.2 Growth and fluorescence measurements .................................................................. 62 3.6 Supplementary materials .................................................................................................. 62 3.7 References .......................................................................................................................... 63 Chapter 4: Molecular toolkit for gene expression control and genome modification in Rhodococcus opacus PD630 ........................................................................................................ 67 4.1 Abstract .............................................................................................................................. 67 4.2 Introduction ....................................................................................................................... 68 4.3 Results and discussion ....................................................................................................... 71 4.3.1 Constitutive promoter library ................................................................................... 71 4.3.2 Heterologous and endogenous plasmid copy numbers ........................................... 72 4.3.3 Antibiotic resistance markers .................................................................................... 75 4.3.4 Genome recombineering ............................................................................................ 77 4.3.5 Neutral integration sites ............................................................................................. 80 4.3.6 CRISPRi-mediated gene repression .......................................................................... 83 4.4 Conclusion .......................................................................................................................... 86 4.5 Materials and methods ...................................................................................................... 87 4.5.1 Strains, plasmids and growth conditions .................................................................. 87 4.5.2 Growth and fluorescence measurements .................................................................. 88 iii 4.5.3 Total DNA extraction and quantitative PCR ........................................................... 89 4.5.4 Identification of neutral sites ....................................................................................