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.

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.

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.

Microorganisms have enormous potential as catalysts for the conversion of renewable organic material into useful chemicals, such as fuels and polymers. But engineering metabolic pathways in microorganisms requires a greater understanding of the enzymes available to catalyze each reaction of a sequential biosynthetic scheme. Fundamental to the construction of organic molecules is the formation of carbon-carbon bonds. This dissertation describes our work to identify and characterize enzymes from non-canonical carbon metabolism pathways in nature. In particular, we focus on two fermentation pathways found in eukaryotic facultative anaerobes: branched acid fermentation in the roundworm Ascaris suum and wax ester fermentation in the single-celled algae Euglena gracilis. These pathways are based on thiolase enzymes, which catalyze carbon-carbon bond formation through Claisen condensation. Our investigation has led to a greater understanding of the factors that determine substrate and product selectivity in thiolases and thiolase-based pathways and has provided insight into the physiology of E. gracilis. We further demonstrate the use of A. suum enzymes to engineer pathways in bacteria for the production of α-methyl organic acids. Our results help to expand the synthetic capabilities of engineered microorganisms.

Fluorination is an important tool for the discovery and production of synthetic pharmaceuticals, as it allows fine-tuning their pharmacokinetic properties and metabolic stability. However, unlike chemists, Nature rarely utilizes fluorine to enhance its molecules, and the few known naturally occurring organofluorines generally lack structural complexity. Despite advances in chemical fluorination methods, the regiospecific fluorination of complex natural products remains a challenge. Thus, we have taken a metabolic engineering approach to develop a novel platform for fluorine incorporation into polyketide natural products. Polyketide natural products are synthesized from one starter unit and multiple acyl-CoA extender units. We designed a modular system in which an inactivating mutation was introduced to the acyltransferase (AT) domain of interest to reduce the native activity of the module, while an alternative fluorine-selective AT enzyme was provided in trans to incorporate fluorine as a fluoromalonyl-CoA extender unit. A malonate transporter and a malonyl-CoA synthetase were introduced to support the intracellular accumulation of fluoromalonate and its enzymatic activation into fluoromalonyl-CoA extender unit, respectively. By engineering a single module construct of 6-deoxyerythronolide B (6dEB) synthase (DEBS), fluorinated triketide lactone was produced as the major polyketide product at mg/L levels in an Escherichia coli host. Extending these engineering concepts to a complete DEBS pathway allowed us to produce novel fluorine-containing 6dEB analogs in E. coli production host in a regiospecific manner. This platform can allow the precise incorporation of fluorine into many positions in the backbones of polyketides. These results open the door to producing a new class of drug-like molecules with potentially diverse and improved properties.

In order to explore new chemical strategies for lignin degradation, we have initiated studies aimed at discovery and characterization of oxidative and accessory enzymes in lignin-reactive soil bacteria that exhibit particularly rich activity towards the depolymerization and utilization of biomass-derived carbon sources. Our interest lies in studying the biochemical logic underlying the transformation of complex substrates by living organisms with the overall goal of elucidating new molecular strategies for lignin degradation. Towards this goal, we began by creating a pipeline for rapid discovery and functional identification of new enzymes from unsequenced bacteria under lignin-reactive growth conditions, an approach that can be applied to any culturable microbe of interest. To do this, we assembled a de novo genome for Amycolatopsis sp. 75iv2 and used this genome sequence to identify genes potentially involved in the degradation system of lignocellulose as well as their downstream degradation products. With a genome sequence now available for proteomics analysis, we analyzed the secretome of A. sp. 75iv2 grown in the presence of lignocellulose by extracellular peroxidase activity of the culture; separation of the secretome by SDS-PAGE and heme-staining revealed the presence of heme-containing proteins which we identified as a catalase-peroxidase and a catalase by LC-MS/MS. Biochemical characterization of the catalase-peroxidase revealed an inability to oxidize non-phenolic ring motifs but showed that phenolic moieties, which encompass ~20% sites in lignin, could be oxidized.

Further analysis of the genome sequence allowed for identification of an interesting and relatively new family of enzymes, the dye decolorizing peroxidases (DyPs). One phylogenetically unique member of this family, DyP2, was heterologously expressed for biochemical and structural characterization. Comparison of the enzymatic activity of this protein indicated that it displayed not only high peroxidase activity against a suite of low- and high-potential DyP substrates, similar to that of fungal DyPs and LiPs, but also an unusually active manganese peroxidase activity akin to that of versatile peroxidases. Furthermore, studies showed DyP2 to have a Mn-dependent oxidase activity that expands its substrate scope to include the oxidation of substrates with non-phenolic aromatic rings. We also solved a crystal structure of DyP2 at 2.25A resolution which revealed the presence of a Mn binding pocket 15 Å from the heme active site.

Finally to begin studying the physiological response of A. sp. 75iv2 to lignin, we developed a set of minimal media conditions that demonstrated A. sp. 75iv2 to be indeed competent to utilize lignin as a sole carbon source. Interestingly, the presence of lignin induced a differential growth response compared to growth on sugar controls. Analysis of the A. sp. 75iv2 secretome further showed the existence of an induction of extracellular peroxidase activity and changes in the secreted heme protein profile. RNA sequencing and proteomics studies were then utilized to profile the global response of A. sp. 75iv2 to lignin and identify candidate proteins and pathways involved in its metabolism. These studies revealed that the dominant response involved a strong activation of Fe assimilation pathways as well as significant upregulation in the expression of universal stress response regulators. Both up- and down-regulation of aromatic degradation pathways were also observed. In order to continue to explore this response, we developed a synthesis for ¹³C-labeled synthetic lignin as a tool for structural studies of lignin by NMR as well as a peroxidase fractionation method as an approach to begin identifying upregulated peroxidases in the A. sp. 75iv2 secretome.

Natural products have played a critical role in various fields for decades. Studying natural product biosynthesis facilitates the discovery of novel bioactive compounds and innovates engineering strategies to create molecules with specific scaffolds or functional groups. In addition to producing tailored molecules, biosynthetic engineering can also be used to achieve mass production of useful compounds. Among all kinds of natural products, terminal alkene and alkyne containing compounds have garnered significant attention due to the reactivity of their terminal functionalities and broad applications in chemical biology research and pharmaceutical industry. Therefore, it is important to understand the biosynthesis of terminal alkene and alkyne containing natural products and engineer pathways to leverage the biosynthetic machinery.Salivabactin is a terminal alkene containing natural product isolated from Streptococcus salivarius K12, which exhibits potent antimicrobial activities towards gram-positive bacteria. Considering its bioactivity and unavailability though synthesis, it is necessary to understand the biosynthesis of salivabactin. Based on enzymatic assays in vitro, we elucidated the biosynthesis and uncovered the minimal set of enzymes required for salivabactin production. Notably, an unprecedented thioesterase involved in the terminal alkene formation was characterized by site-directed mutagenesis and structural elucidation. With the understanding of biosynthesis, we were able to engineer the pathway to increase compound titer. Through heterologous expression and growth optimization in E. coli, we achieved a remarkable seven-fold enhancement in titer compared to that in the native host. This work broadens our understanding of terminal alkene biosynthesis and facilitates the mass production of new antibiotics to combat emerging multi-drug resistance. In addition to terminal alkene containing natural products, there are also diverse natural products with the terminal alkyne moiety, formed through various biosynthetic mechanisms. Among them, we focused on the family of membrane-bound desaturases/acetylenases, represented by JamB and TtuB. These enzymes are capable of catalyzing the terminal alkyne formation on acyl carrier protein (ACP)-tethered substrates. Intriguing mechanistic questions surrounding JamB and TtuB prompted the biochemical and structural studies, necessitating solubilized and active forms of these membrane-bound enzymes. To achieve this, we employed different strategies, including residue swapping, cell-free protein synthesis and lipoprotein shielding. Our efforts yielded promising results in terms of solubilization and enzyme activity, paving the way for further mechanistic exploration into terminal alkyne biosynthesis. Meanwhile, with the successful engineering to generate terminal alkyne tagged polyketides, we extended the applicability of terminal alkyne tagging to lipopeptides, as demonstrated by two NRPS systems in vivo and in vitro. Notably, we employed an innovative engineering strategy involving a non-elongating ketosynthase domain, which facilitated the transfer of acyl chains between ACPs. This establishes a versatile method for incorporating non-cognate ACPs into assembly lines.