Cirino, Patrick C.2024-01-27December 22023-12Portions of this document appear in: Wang, Yixi, Nam Nguyen, Seung H. Lee, Qinxuan Wang, Jeremy A. May, Ramon Gonzalez, and Patrick C. Cirino. "Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1‐methylalkyl) succinates." Biotechnology and Bioengineering 119, no. 1 (2022): 315-320.https://hdl.handle.net/10657/16222Rising global energy demand and geopolitical tensions have renewed interest in expanding domestic production of natural gas liquids (NGLs) like propane and butane. These alkanes possess high energy density but present transport challenges over long distances. Industrial bioconversion of NGLs into liquid fuels using traditional processes like Fischer-Tropsch (FT) is capital intensive. As an alternative, certain microbes can enzymatically activate alkanes with methyl-alkylsuccinate synthase (MAS), enabling more economical bioprocessing. However, native MAS-containing bacteria have limited genetic tractability. A majority of the research described in this dissertation focused on establishing functional MAS expression systems in the well-studied host Escherichia coli. Mas genes from anaerobic bacteria were heterologously expressed in E. coli under strict anaerobic conditions to maintain activity. Systematic optimization of media composition, cofactors, and culturing conditions improved alkylsuccinate production from 50μM to 200μM. Bioconversion of propane, butane, and hexane by the recombinant MAS was confirmed via GC-MS. Characterization of the MAS complex revealed a non-essential subunit, enabling comparison to the related benzylsuccinate synthase. Additionally, E. coli strains were engineered to enhance interactions with hydrophobic substrate. Surface proteins including fimbriae, flagella, and curli were engineered for inducible and tunable expression. Optimizing expression levels prevented toxicity while improving interactions with alkane emulsions and biofilm formation. In summary, this dissertation establishes the foundation for engineering E. coli capable of activating SCAs with enhanced interactions to hydrophobic substrates.application/pdfengThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).E. coli, bioremediation, alkane2024-01-27Thesisborn digital