Amino acids are one of nature’s privileged scaffolds for chemical and biomolecular diversification. Beyond the standard twenty amino acids used in protein synthesis, hundreds more noncanonical amino acids (ncAAs) have been found in natural products with useful chemical features. Here we present a resistance-guided approach to discovering ncAA natural products with an emerging class of enzymes: tRNA-deacylases (tRDs). Beginning with the discovery and characterization of a tRD with specificity towards a ncAA, we performed family-wide analyses of tRDs in biosynthetic gene clusters (BGCs) and established tRDs as a widely present genomic marker of ncAA metabolism. These efforts enabled us to discover and elucidate biosynthetic pathways for several new ncAAs and natural products. Through strategies like protein engineering and cascade design, we employed several of these enzymes as biocatalysts to achieve transformations that have been challenging to access otherwise. We anticipate the advances in genome mining and experimental characterization developed in this work will have broad applications for discovering new natural products with ncAAs and useful biocatalysts.

The steep environmental costs of petrochemical-derived products have created a pressing need for sustainable approaches to chemical synthesis. To address this need, scientists and engineers have developed diverse methods for capturing and either storing or using greenhouse gases to make value-added chemicals. In this effort, many scientists have looked to Nature for inspiration. Nature has evolved many methods for capturing CO2 and using it as a carbon source for autotrophic growth. Of all the diverse forms of autotrophy, chemoautotropic metabolism is particularly remarkable for its versatile use of inorganic electron donors. If this versatility could be harnessed and coupled to sustainable sources of electrons and energy, chemoautotrophs may be used as a metabolic host for sustainable chemical synthesis.

This thesis explores two approaches for the engineering of chemoautotrophic metabolism for the production of value-added chemicals. In the first approach, essential genes for the Wood-Ljungdahl carbon fixation pathway were investigated in Moorella thermoacetica, a model acetogenic bacteria. A panel of gene candidates were identified for the appropriate maturation of the acetyl-CoA synthase/CO dehydrogenase (ACS/CODH) complex, an important metalloenzyme complex in the Wood-Ljungdahl pathway. The most promising candidate, AcsF, was recombinantly expressed with ACS in Escherichia coli, showing an increase in ACS nickel content. Furthermore, methods were developed to produce knockouts in M. thermoacetica to assess the native role of our gene candidates and measure the in vitro and in vivo activity of a recombinant ACS/CODH complex from E.coli.

In our second approach, we attempted to engineer Methanococcus maripaludis, a model methanogenic archaea, to produce 3-hydroxybutyrate (3HB) and polyhydroxybutyrate (PHB). NAD(H) pools in M. maripaludis were less than 5% of the pool in anaerobically-grown E.coli, suggesting that low NADH availability may be a metabolic bottleneck for biosynthesis. Using transcriptomic and proteomic studies, alanine dehydrogenase was identified as a critical native enzyme for in vivo NADH regeneration and improvements in product titers were observed in alanine-dependent growth. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of NAD+-dependent formate dehydrogenase from Candida boidinii and a synthetic nicotinamide salvage pathway. Engineered strains of M. maripaludis with improved NADH pools produced up to 150 ± 4 mg/L of PHB in McNA medium, a more than 2.5-fold improvement over the starting strain.

To further characterize heterologous gene expression, native transcript structure in M. maripaludis was more carefully analyzed. Methanogenic archaea display characteristics of translation that are analogous to both eukaryotic and prokaryotic processes, but some features remain to be determined. To address this, we conducted a transcriptome assembly and determined the consensus Shine-Dalgarno (SD) sequence of GGAGGA from our 5’ UTR-containing sequences. Our predicted transcripts were verified using 3’ UTR sequencing. In addition, we found that a large fraction of our transcripts were leaderless, motivating us to investigate translation initiation for both leaderless and leadered transcripts. To probe this question, we developed a ribosome profiling methodology that could be used to precisely identify the ribosome’s position and interactions with mRNA. These developments help pave the way towards a robust heterologous gene pathway design strategy for this species. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.

Magnetotactic bacteria have evolved complex subcellular machinery to construct linear chains of magnetite nanocrystals that allow the host cell to sense direction. Each mixed-valent iron nanoparticle is mineralized from soluble iron within a membrane-encapsulated vesicle termed the magnetosome, which serves as a specialized compartment that regulates the iron, redox, and pH environment of the growing mineral. In order to dissect the biological components that control this process, we have carried out genetic and biochemical studies of proteins proposed to function in iron mineralization in Magnetospirillum magneticum AMB-1. As iron biomineralization by magnetotactic bacteria represents a particularly interesting case for understanding how the production of nanomaterials can be programmed at the genetic level, we also apply synthetic biology techniques towards the production of new cellular materials and new cellular functions.

As the production of magnetite requires both the formation of Fe(II) and Fe(III), the redox components of the magnetosome play an essential role in this process. Using genetic complementation studies, we show that the redox cofactors or heme sites of the two putative redox partners, MamP and MamT, are required for magnetite biomineralization in vivo and that removal of one or both sites leads to defects in mineralization. We develop and optimize a heterologous expression method in the E. coli periplasm to cleanly isolate fully heme-loaded MamP for biochemical studies. Spectrochemical redox titrations show that the reduction potential of MamP lies in a different range than other c-type cytochrome involved in either Fe(III) reduction or Fe(II) oxidation. Nonetheless, in vitro mineralization studies with MamP and Fe(II) show that it is able to catalyze the formation of mixed-valent Fe(II)/Fe(III) oxides such as green rust.

Biomineralization also requires lattice-templating proteins that guide the growth of the functional crystalline material. We use in vitro binding and synthesis studies with putative magnetite-templating proteins, the Mms6 family proteins, to show that they are competent to bind and stabilize non-thermodynamically stable faces of magnetite. We also use in vitro iron mineralization to show that the Mms6 family proteins can work together with the redox protein MamP to produce mixed-valent iron oxides from soluble Fe(II) species and to control mineral structure. Further studies with Mms7ct indicate that it and other Mms6 family proteins may play a more significant role in controlling magnetite mineral structure than previously hypothesized. Beyond simple control of size and shape of magnetite, it may also template the crystal lattice of the mineral itself similar to what has been observed with calcium biomineralization, where unstable crystal forms and phases of the mineral are stabilized by interaction with peptides and other macromolecules.

We next set up and begin testing systems to engineer magnetotactic bacteria for the production of new functional materials. We replace the metal-binding C-terminus of native Mms6 family proteins in AMB-1 with peptides known to precipitate metal oxides in vitro under mild conditions. Initial characterization of the behavior of these constructs in AMB-1 has been carried out, although additional experiments are required to test whether they can enable formation of new materials in vivo. We also discuss developing a cellular biosensor based on the formation of a magnetic material in response to an analyte.

Macrolide natural products have been a rich source of medicinally relevant molecules. Macrolides are assembled by large protein complexes known as polyketide synthases (PKSs) via polymerization reactions of simple acetate and propionate derived building blocks. While these enzymes are capable of generating molecules of impressive structural diversity, the introduction of nonnative and potentially bioactive functional groups into the backbone of these molecules remains challenging and is mostly limited to synthetic methods. The incorporation of fluorine atoms or fluorinated moieties into both synthetic and semi-synthetic medicinally relevant molecules has been shown to improve their bioactivity, bioavailability, and stability. However, the very properties that make fluorine a valuable component of pharmaceuticals also makes it challenging to incorporate via synthetic methods. Seeking to capitalize on the modular nature of PKSs, previous work in our lab has shown that fluorinated polyketides can be synthesized by using fluorinated analogs of PKS building blocks. The work described in this dissertation follows a mechanistic approach to fluorinated extender unit incorporation into polyketides. By examining the nature of fluorinated extender unit incorporation into the 6-deoxyerythronolide B synthase modules, we were able to build a platform showing suitable promise for the site-specific incorporation of fluorine into the backbone of medicinally relevant natural products.

The soil-dwelling bacterium Streptomyces cattleya produces the antibiotics fluoroacetate and fluorothreonine, and has served as a model system for the discovery of naturally occurring fluorine-selective biochemistry. While fluoroacetate has long been known to act as an inhibitor of the TCA cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, I show that while fluorothreonine is a substrate for translation, this activity is averted in S. cattleya by the activity of two conserved proteins. The first, SCAT_p0564, acts in vitro and in vivo as a fluorothreonyl-tRNA selective hydrolase, while the second, SCAT_p0565, is proposed to be a fluorothreonine exporter. Additionally, overexpression of SCAT_p0564 in the model strain Streptomyces coelicolor M1152 confers resistance to fluorothreonine, suggesting that the antibiotic activity of this compound is related in part to its ability to enter the proteome. The ability of SCAT_p0564 to selectively hydrolyze fluorothreonyl- over threonyl-tRNA is striking, given that these macromolecular substrates differ by a single atom. In order to understand the basis of this selectivity, I have solved the crystal structure of this enzyme, and characterized its ability to act on the related substrate chlorothreonyl-tRNA. I also have also begun to elucidate the regulatory architecture of the organofluorine biosynthesis locus, with the aim of understanding how this unusual process is controlled. I find that transcription of the fluorinase is driven by the master regulator FlG, while FlF is an amino acid binding transcription factor that may be required for full expression of the fluorothreonine transaldolase. These findings expand the range of known naturally occurring fluorine biochemistry, and a represent a step towards the rational discovery of new organofluorine metabolism in nature.

The development of sustainable synthesis methods for societally important chemical products which serve as fuels, industrial feedstocks and materials is a pressing global challenge. Many of these target molecules are currently produced from fossil fuel-derived starting materials, which is problematic due both to the impending exhaustion of these resources and the dire environmental consequences of unrestrained carbon dioxide release into the atmosphere. An attractive alternative is using carbon dioxide along with sustainable energy sources for the production of these chemical targets. This thesis explores the development of multiple sustainable synthesis platforms based on interfacing inorganic materials, which function well for solar energy capture and charge transfer, with autotrophic microorganisms, which serve as biocatalysts, able to fix carbon dioxide into value-added products using the energy provided by the inorganic components.

An initial approach focused on the development of a hybrid bioinorganic device in which sustainable electrical and/or solar input drove production of hydrogen gas from water splitting using biocompatible inorganic catalysts. The hydrogen was then used by autotrphic microorganisms as a source of reducing equivalents for conversion of CO2 to the value-added chemical product methane. Employing platinum or a newly synthesized earth-abundant substitute, α-NiS, as biocompatible hydrogen evolution reaction (HER) electrocatalysts and Methanosarcina barkeri as a biocatalyst for CO2 fixation, robust and efficient electrochemical CO2 to CH4 conversion was demonstrated at up to 86% overall Faradaic efficiency for ≥7 d. Introduction of indium phosphide photocathodes and titanium dioxide photoanodes afforded a fully solar-driven system for methane generation from water and CO2, establishing that compatible inorganic and biological components can synergistically couple light-harvesting and catalytic functions for solar-to-chemical conversion.

A second device attempted to mimic the biosynthetic process of natural photosynthesis, where solar energy powers the reduction of CO2 to common biochemical building blocks, which are subsequently used for the synthesis of the complex mixture of molecular products that form biomass. The artificial photosynthesis system described here functions via a similar two-step process: A biocompatible high-surface-area silicon nanowire array harvests light energy to provide reducing equivalents to the obligately anaerobic acetogen, Sporomusa ovata, for the photoelectrochemical production of acetic acid with low overpotential (η < 200 mV), high Faradaic efficiency (up to 90%), and long-term stability (up to 200 h). The resulting acetate (∼6 g/L) can be activated to acetyl coenzyme A (acetyl-CoA) by strains of genetically engineered Escherichia coli and used as a building block for a variety of value-added chemicals, such as n-butanol, polyhydroxybutyrate (PHB) polymer, and three different isoprenoid natural products. Due to the local chemical environment within the nanowire array, the systems was also shown to perform well under aerobic conditions (21% O2), allowing the cultivation of an obligate anaerobe using oxygen-containing gas feedstocks for growth. This system demonstrated the power of interfacing biocompatible solid-state nanodevices with living systems in order to create programmable platforms for chemical synthesis entirely powered by sunlight.

Informed by the successes of the previous systems, a next generation device was envisioned in which the autotrophic microorganism, which was interfaced with the biocompatible inorganic components, is directly engineered to produce value-added products, offering the simplicity of a single organism system with the product scope of co-culture platforms. Towards this goal methods for metabolic engineering of Methanococcus maripaludis, a methanogenic archaea, have been developed. These efforts have chiefly focused on achieving robust expression of multienzyme heterologous pathways, a goal complicated by the incompletely understood translational machinery in this organism. For optimization the 3-hydroxybutyrate pathway was used, which results in production of a monomer unit, which can be polymerized into the bioplastic PHB. Robust expression of the full 3-hydroxybutyrate biosynthetic pathway was achieved using a strong Methanosarcina barkeri promoter. Activity of all pathway enzymes was demonstrated in cell lysates; however, no product formation was observed. It is currently hypothesized that this is due to a lack of appropriate redox cofactors, specifically NADH. The NADH pool observed in M. maripaludis was found only to be 3% of that observed in E. coli. Current efforts are underway to enlarge this cofactor pool and incorporate it into the redox metabolism of the organism.

This work has demonstrated the power of hybrid biological-inorganic devices for the sustainable synthesis of value-added products. Systems have been developed that are fueled using sustainable sources of electric current or directly by illumination. A variety of product has been produced, both native products as well as more complex products, using a co-culture system. Efforts are ongoing to develop metabolic engineering tools for the unique class of organisms amenable to integration with inorganic materials. These engineering efforts will pave the way for future simplified devices with expanded product scopes.

Fluorine is a critically important element for the design of safe and effective drugs in the modern pharmaceutical industry. As the field of synthetic methodology for fluorination of organic compounds has flourished, biocatalysis has also been rapidly adopted as a means to achieve high reaction selectivity and process sustainability. However, the areas of biocatalysis and fluorine chemistry have rarely intersected directly, owing to the rarity of fluorine-processing enzymes in Nature. We envision that this methodology gap can be bridged by the action of carbon-carbon bond forming enzymes upon non-native fluorinated substrates, thus transforming simple organofluorine building blocks into more complex organofluorines. In this dissertation, we describe the application of enzymatic aldol addition to fluoro-aldol reactions that furnish value-added organofluorine products bearing fluorine stereocenters. Our development of a novel platform for biocatalytic organofluorine synthesis began with the discovery that Type II pyruvate aldolases of the HpcH family use fluoropyruvate as an alternative nucleophilic substrate. After studying the stereoselectivity, kinetics, and mechanism of the fluoropyruvate aldol reaction, the afforded products were converted to diversely functionalized organofluorines through downstream reactions. Next, the HpcH system was expanded beyond fluoropyruvate to generalized β-fluoro-α-ketoacids as nucleophiles. The investigation of these challenging substrates, aided by rational active site engineering, resulted in the first synthesis of tertiary fluorides via biocatalytic C-C bond formation. Usefulness of the HpcH platform was demonstrated with preparative syntheses of products relevant to drug fragments and bioactive natural products. Finally, we studied Type I aldolases with the unique ability to use simple aldehydes and ketones as nucleophiles. Their activity on fluorinated ketones may enable the efficient synthesis of fluorinated sugars, which are applicable to pharmaceuticals and chemical biology. Taken together, this dissertation delineates a selective and sustainable biocatalytic approach to access the unique properties conferred by fluorination, which may have broad implications for improved process design towards important organofluorines.

Living systems are remarkably efficient in their ability to build complex molecular function, using a relatively small set of chemical functional groups to drive the wide range of life processes. In contrast, synthetic compounds access a much broader scope of structural and reaction diversity to tune desired chemical properties. However, unusual functional groups can be found in Nature. Many pathways for secondary metabolism produce compounds that could be useful for chemical applications if their biosynthesis could be repurposed. Here, we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid, β- ethynylserine, by the microbe Streptomyces cattleya through an unexpected reaction sequence that includes radical halogenation and oxidative C-C bond cleavage, followed by a desaturation that proceeds through a putative allene intermediate. We also report progress in porting the bes pathway into heterologous host, particularly E. coli. Furthermore, we present preliminary work on the discovery of a protein who’s function is to prevent translation of β-ethynylserine by acting as a tRNA proofreading enzyme. This pathway, and the auxiliary proteins related to control of translation of non-standard amino acids, offers the potential to genetically encode the de novo production of halo-, alkene-, and alkyne-labeled proteins and peptides from glucose to provide bioorthogonal functionality for downstream applications.

Natural products use exquisite structural complexity to tailor a limited repertoire of functional groups for different molecular functions. These molecules have been adopted or adapted by humans to create a large fraction of current therapeutics. However, those adaptations have not always been able to combine the chemical diversity possible for totally synthetic compounds with the many advantages of biosynthesis. Extremely rare in nature but ubiquitous in synthetic pharmaceuticals, fluorine is a unique tool for molecular design, and an excellent candidate for expanding biosynthetic diversity. This dissertation describes our work to enable efficient fluorine incorporation into complex biosynthetic pathways, both in vitro and in the context of a whole organism, and our progress in enabling fluorinated polyketide biosynthesis. We hope to set the stage for a dramatic expansion in polyketide structural diversity via engineered biosynthesis, using a design element that could improve bioactive properties and furnish new therapeutics.

The biological diversity found throughout the world contains equally wondrous chemical diversity that can operate with the precision, efficiency, and scale that humanity has yet to attain. This capacity is an untapped resource that must be understood and harnessed to address pressing global needs for food, energy, medicine, and materials. Wielding this power will require a deeper understanding of how a given biological process occurs in the context of a cell. Metabolic pathways are an ideal model system to study biochemical processes in vivo as they are integral to the cell’s survival, they are regulated on multiple interlocking levels, and they have a broad dynamic range with many measurable inputs and outputs.

We have studied a synthetic metabolic pathway in E. coli as a means of gaining insight into biological regulatory networks, but also with the goal of optimizing production of the second-generation biofuel n-butanol. Our previous pathway suffered from poor substrate specificity in the final enzyme, leading to off-target products and decreased yield. This enzyme, AdhE2, is a bifunctional aldehyde alcohol dehydrogenase that catalyzes sequential reductions of acyl-CoAs to alcohols through aldehyde intermediates. The enzyme was biochemically characterized to determine its substrate specificity, coordination between active sites, and oligomerization behavior. The enzyme was found to be undesirable for butanol production and new classes of enzymes were explored.

To replace AdhE2 we employed bioinformatic methods to identify a family of monofunctional aldehyde dehydrogenases. This family was screened and a highly specific enzyme was identified. The improved butanol production pathway was then a suitable tool for exploring regulatory mechanisms controlling metabolism by employing whole genome mutagenesis and selection. A butanol production strain was engineered such that its growth under anaerobic conditions was directly linked to butanol production. This strain’s genome was mutagenized and subjected to anaerobic growth selection to enrich for mutants producing elevated levels of butanol. We then sequenced the genomes of these strains to identify regulatory mechanisms impacting butanol production.

Finally, we expanded upon our butanol production pathway by leveraging the previously identified aldehyde dehydrogenase family for the production of the commodity chemicals 1,3-butanediol and 4-hydroxy-2-butanone. Aldehyde and alcohol dehydrogenases were identified by a variety of methods and screened for production. We developed several strategies to afford control over the ratio of products produced including pathway design and expression level tuning. Directed evolution methods including DNA shuffling and saturation mutagenesis were also used to further tailor aldehyde dehydrogenases for the desired products.

In sum we have extensively characterized a number of aldehyde and alcohol dehydrogenases from multiple families. Optimized pathways for production of n-butanol, 1,3-butanediol, and 4-hydroxy-2-butanone were developed. A genetic selection for metabolite production was developed and validated, and evolved strains were characterized to identify important regulatory mechanisms.