UC Santa Barbara

Synthetic biology holds promise for adapting and modifying molecular systems found in nature toward improving human life. This has sparked a modern bioindustry with applications ranging from biomanufacturing pharmaceutical products to engineered microbes for supporting space exploration. In the last decade two biotechnological advances have revolutionized the field of synthetic biology: CRISPR/Cas-mediated genome editing and directed protein evolution. As these principles have matured, advances in automation and artificial intelligence have been quickly adopted as major tools toward the generation of new molecular biotechnologies.

This thesis incorporates the use of specific genetic systems and broad principles of evolution toward engineering new biotechnologies. To adopt novel genetic systems in synthetic biology applications they must first be fully understood. Thus, an important interest underlying the applied aspect of synthetic biology is solving genetic and proteomic mechanisms of action through basic science. One way of doing so is to understand the effects that these genetic systems of interest have had on the natural evolution of the organisms that utilize them.

Toward this interest, my first chapter aims to better understand the impact that diversity generating retroelements (DGRs), may have had on evolving vast morphological, physiological, or adaptive complexity observed within the cyanobacterial phylum. This first chapter develops the hypothesis that, in cyanobacteria, DGRs have evolved to accelerate the evolution of proteins of a distinct functional class, compared to the DGRs of all other prokaryotic organisms currently known. This further sets apart cyanobacteria as an incredibly unique and diverse taxonomic clade within bacteria and sets DGRs apart as an important player in the evolutionary success of cyanobacteria. A paper has been communicated on the work presented in my first chapter, detailed below.

Aside from the potential evolutionary and ecological impact DGRs may have had within the cyanobacterial phylum they are also of interest for biotechnological applications. DGRs are a potential tool for continuous in-vivo, targeted hyper-diversification within a small region of a gene. This is a desirable tool for directed evolution efforts, as the field currently lacks a means for generating highly diverse random mutations in-vivo in targeted genomic loci. As such, in-vivo directed evolution schemes are generally not attempted, though the efficiency of in-vivo natural selection schemes may be much greater when compared to in-vitro screening of individual protein variants as a selection scheme.

The remaining two chapters of my thesis deal with mining the marine environment for other molecular systems that hold significant promise for developing new biotechnologies including products that may aid in space exploration. Marine isolates of Bacillus subtilis have been shown to display a unique propensity for producing highly complex and highly bioactive secondary metabolites. Furthermore, their ability to form endospores that are highly resistant to extreme environmental conditions make them an ideal candidate for engineering toward bioremediation and drug production for space conditions.

The majority of pharmaceuticals are set, conservatively, to expire within two years of the manufacturing date, after which the reported potency and pharmacokinetics are no longer guaranteed. Though there are efforts to study and extend pharmaceutical expiration dates, viability of a product will vary based on manufacturing lot, storage conditions, and packaging. The environs of spaceflight may also affect the decay of pharmaceuticals, though the extent remains unclear. As such, for longer crewed missions, the pharmaceutical supply at launch is unlikely to remain viable for the duration of the mission. However, replenishment becomes complicated on any space mission, and particularly on longer- duration missions, when resupply is difficult, and it is impossible to predict the specific medication needs of the crew. Thus, a platform for on-demand drug synthesis is necessary to ensure the success of long-term space exploration and colonization missions to Mars and beyond.

Thus, my second chapter presents the first step in a three-step process known as an “Astropharmacy”, a peptide drug production platform that uses genetically engineered bacterial spores or a cell-free system to produce drugs on-demand. Not only would this platform allow astronauts to produce drugs according to their needs, but it would also have utility on Earth, with applications including bioterror prevention, orphan drug production, and drug production in remote areas where refrigeration is impossible. Whereas my second chapter deals with mining the marine environment for microbial hosts which have unique and favorable evolutionary characteristics for space, my third chapter seeks to isolate a microalgal marine protein and study its ability to be engineered further to meet different needs we may have on earth.

Microalgae have been considered the most promising biological chemists for the mass production of biologically sourced fuels. Algal organisms may have adapted this ability as a way of regulating cell membrane curvature and hydrophobicity. One goal in biofuel-focused synthetic biology is to reverse-engineer pathways that lead to the production of different hydrocarbon molecules to increase the efficiencies, yields, and culture survivability. One of the newest discoveries that apply to this effort is fatty acid photodecarboxylase (FAP) – an enzyme which uses light to make fuel from acidic fats.

My third chapter details the need for confirming homologs of the originally discovered enzyme. Can mutations that work in the original enzyme be successfully transferred to its homologs? If so, it would allow us to consider homologs for directed evolution strategies including chimeric gene splicing. It would also allow us to add molecular tools found in other organisms to the biocatalytic toolbox of enzymes that enable light-driven decarboxylation of fatty acid substrates.