Genome Mining, Biosynthesis and Catalysis of a Novel Fungal Polyketide Synthase-Carnitine Acyltransferase Fusion Enzyme

This dissertation describes the genome mining, biosynthesis, and catalysis of a novel fungal polyketide synthase-carnitine acyltransferase fusion enzyme. Fungal highly-reducing polyketide synthases (HRPKS) catalyze the biosynthesis of polyketide natural products such as the cholesterol-lowering drug, lovastatin. Investigation into unexplored families of HRPKS enzymes such as the HRPKS-cAT fusion enzymes provides opportunities to discover new drug leads. This research will also improve our understanding of HRPKS enzymatic catalysis for future engineering purposes.Chapter 1 introduces the function, iterative catalysis, and biosynthetic framework of the fungal HRPKS enzymes. Partnering oxygenation enzymes which functionalize the polyketide scaffold are also assessed to emphasize the meticulous synchronization involved in HRPKS biosynthesis. Several examples of HRPKS biosynthesis are provided to illustrate the coordination required for the formation of the final natural product.Chapter 2 examines the genome mining of HRPKS-cAT enzymes and highlights their differences from canonical HRPKS catalysis. The HRPKS-cAT enzyme, Tv6-931, is reconstituted and the biosynthesis of polyketide-polyol conjugates bearing a rare gem-dimethyl moiety is discussed. Based on enzymatic assays using the Tv6-931 whole enzyme and dissected enzyme variants, an unusual polyketide “recapture” (oxyester-thioester transacylation) is proposed to explain the formation of the gem-dimethyl moiety.Chapter 3 investigates the biosynthetic, polyketide “recapture” mechanism introduced in Chapter 2. Enzyme kinetic studies show that the cofactor-independent, polyketide recapture is necessary for the gem-dimethyl adduct to be the major product. Based on the oxyester-thioester transacylation mechanism (recapture), a chemoenzymatic strategy was developed to conjugate other nucleophiles to create a variety of polyketide oxyester, thioester and amide adducts. A one-pot, multi-enzyme approach was used to demonstrate polyketide functionalization using HRPKS-cAT enzymes for proof-of-principle

Genome Mining, Biosynthesis and Catalysis of a Novel Fungal Polyketide Synthase-Carnitine Acyltransferase Fusion Enzyme

This dissertation describes the genome mining, biosynthesis, and catalysis of a novel fungal polyketide synthase-carnitine acyltransferase fusion enzyme. Fungal highly-reducing polyketide synthases (HRPKS) catalyze the biosynthesis of polyketide natural products such as the cholesterol-lowering drug, lovastatin. Investigation into unexplored families of HRPKS enzymes such as the HRPKS-cAT fusion enzymes provides opportunities to discover new drug leads. This research will also improve our understanding of HRPKS enzymatic catalysis for future engineering purposes.Chapter 1 introduces the function, iterative catalysis, and biosynthetic framework of the fungal HRPKS enzymes. Partnering oxygenation enzymes which functionalize the polyketide scaffold are also assessed to emphasize the meticulous synchronization involved in HRPKS biosynthesis. Several examples of HRPKS biosynthesis are provided to illustrate the coordination required for the formation of the final natural product.Chapter 2 examines the genome mining of HRPKS-cAT enzymes and highlights their differences from canonical HRPKS catalysis. The HRPKS-cAT enzyme, Tv6-931, is reconstituted and the biosynthesis of polyketide-polyol conjugates bearing a rare gem-dimethyl moiety is discussed. Based on enzymatic assays using the Tv6-931 whole enzyme and dissected enzyme variants, an unusual polyketide “recapture” (oxyester-thioester transacylation) is proposed to explain the formation of the gem-dimethyl moiety.Chapter 3 investigates the biosynthetic, polyketide “recapture” mechanism introduced in Chapter 2. Enzyme kinetic studies show that the cofactor-independent, polyketide recapture is necessary for the gem-dimethyl adduct to be the major product. Based on the oxyester-thioester transacylation mechanism (recapture), a chemoenzymatic strategy was developed to conjugate other nucleophiles to create a variety of polyketide oxyester, thioester and amide adducts. A one-pot, multi-enzyme approach was used to demonstrate polyketide functionalization using HRPKS-cAT enzymes for proof-of-principle

Development of a polylactic acid (PLA) polymer with an acid-sensitive N-ethoxybenzylimidazole (NEBI) crosslinker as a drug delivery system

This thesis describes the development of an alkyne- functionalized poly-lactic acid (PLA) polymer with an acid -sensitive N-ethoxybenzylimidazole (NEBI) linker conjugated to doxorubicin as a drug delivery system (DDS). It also entails the specific qualities of the drug delivery system as a chemotherapeutic regiment. Chapter 1 introduces why drug delivery systems are needed and their desirable properties. In particularly, chapter 1 discusses the roles of [polymeric] carriers and linkers in drug delivery. It also covers some of the drug-delivery strategies specific to chemotherapy. Chapter 2 presents the synthetic route and methodology to produce functionalized PLA, specifically with terminal alkyne moieties. It also covers different strategies to synthesize lactide derivatives with different functional groups. Chapter 3 introduces the synthetic scheme to producing bi-functional N-ethoxybenzylimidazole (NEBI) derivatives. These novel linkers previously displayed accelerated rates of hydrolysis at lower pH. NEBI derivatives also serve as acid-sensitive linkers in the synthesis of my drug delivery system. Chapter 4 discusses the characteristics of my drug delivery system including incorporation percentages, payload, hydrolysis rate and cytotoxicity. The future directions of this project are also elaborated on in that chapte

Development of a polylactic acid (PLA) polymer with an acid-sensitive N-ethoxybenzylimidazole (NEBI) crosslinker as a drug delivery system

This thesis describes the development of an alkyne- functionalized poly-lactic acid (PLA) polymer with an acid -sensitive N-ethoxybenzylimidazole (NEBI) linker conjugated to doxorubicin as a drug delivery system (DDS). It also entails the specific qualities of the drug delivery system as a chemotherapeutic regiment. Chapter 1 introduces why drug delivery systems are needed and their desirable properties. In particularly, chapter 1 discusses the roles of [polymeric] carriers and linkers in drug delivery. It also covers some of the drug-delivery strategies specific to chemotherapy. Chapter 2 presents the synthetic route and methodology to produce functionalized PLA, specifically with terminal alkyne moieties. It also covers different strategies to synthesize lactide derivatives with different functional groups. Chapter 3 introduces the synthetic scheme to producing bi-functional N-ethoxybenzylimidazole (NEBI) derivatives. These novel linkers previously displayed accelerated rates of hydrolysis at lower pH. NEBI derivatives also serve as acid-sensitive linkers in the synthesis of my drug delivery system. Chapter 4 discusses the characteristics of my drug delivery system including incorporation percentages, payload, hydrolysis rate and cytotoxicity. The future directions of this project are also elaborated on in that chapte

Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity.

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays

Biosynthesis of Strained Piperazine Alkaloids: Uncovering the Concise Pathway of Herquline A

Nature synthesizes many strained natural products that have diverse biological activities. Uncovering these biosynthetic pathways may lead to biomimetic strategies for organic synthesis of such compounds. In this work, we elucidated the concise biosynthetic pathway of herquline A, a highly strained and reduced fungal piperazine alkaloid. The pathway builds on a nonribosomal peptide synthetase derived dityrosine piperazine intermediate. Following enzymatic reduction of the P450-cross-linked dicyclohexadienone, N-methylation of the piperazine serves as a trigger that leads to a cascade of stereoselective and nonenzymatic transformations. Computational analysis of key steps in the pathway rationalizes the observed reactivities

Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a <i>C</i>-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 10³ times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays

Biosynthesis of Strained Piperazine Alkaloids: Uncovering the Concise Pathway of Herquline A

Nature synthesizes many strained natural products that have diverse biological activities. Uncovering these biosynthetic pathways may lead to biomimetic strategies for organic synthesis of such compounds. In this work, we elucidated the concise biosynthetic pathway of herquline A, a highly strained and reduced fungal piperazine alkaloid. The pathway builds on a nonribosomal peptide synthetase derived dityrosine piperazine intermediate. Following enzymatic reduction of the P450-cross-linked dicyclohexadienone, <i>N-</i>methylation of the piperazine serves as a trigger that leads to a cascade of stereoselective and nonenzymatic transformations. Computational analysis of key steps in the pathway rationalizes the observed reactivities

Phenalenone Polyketide Cyclization Catalyzed by Fungal Polyketide Synthase and Flavin-Dependent Monooxygenase

Phenalenones are polyketide natural products that display diverse structures and biological activities. The core of phenalenones is a peri-fused tricyclic ring system cyclized from a linear polyketide precursor via an unresolved mechanism. Toward understanding the unusual cyclization steps, the <i>phn</i> biosynthetic gene cluster responsible for herqueinone biosynthesis was identified from the genome of Penicillium herquei. A nonreducing polyketide synthase (NR-PKS) PhnA was shown to synthesize the heptaketide backbone and cyclize it into the angular, hemiketal-containing naphtho-γ-pyrone prephenalenone. The product template (PT) domain of PhnA catalyzes only the C4–C9 aldol condensation, which is unprecedented among known PT domains. The transformation of prephenalenone to phenalenone requires an FAD-dependent monooxygenase (FMO) PhnB, which catalyzes the C2 aromatic hydroxylation of prephenalenone and ring opening of the γ-pyrone ring simultaneously. Density functional theory calculations provide insights into why the hydroxylated intermediate undergoes an aldol-like phenoxide–ketone cyclization to yield the phenalenone core. This study therefore unveiled new routes and biocatalysts for polyketide cyclization