This dissertation is focused on exploring the potential of bacteria for the biosynthesis of natural products with the purposes of generating novel natural product derivatives and of improving the titer of pharmaceutically important natural products. A wide variety of compounds from various sources have been historically used in the treatment and prevention of diseases. Natural products as a major source of new drugs are extensively explored due to their huge structural diversity and promising biological activities such as antimicrobial, anticancer, antifungal, antiviral and antioxidant properties. For instance, penicillin as an early-discovered antimicrobial agent has saved millions of lives, indicating the historical importance of natural products. However, the alarming rise in the prevalence of drug resistance is a serious threat to public health and it has coincided with the decreasing supply of new antibiotics. Bacteria with a tremendous undiscovered potential have still been one of the richest sources of bioactive compounds to tackle the growing threat of antibiotic-resistant pathogens. Nevertheless, the production level of those important compounds is often quite low, and often undetectable using current analytical techniques. To expand the chemical repertoire of nature and to increase the titer of the natural products, researchers have developed various strategies, such as heterologous expression, co-cultivation of different bacteria, optimization of fermentation conditions, discovery of new species, engineering of biosynthetic enzymes, and manipulating regulatory elements. Thus, in my dissertation research, I have exploited a few of these strategies. First, I heterologously expressed some of the biosynthetic genes from the sch biosynthetic gene cluster, resulted in the production of a novel glycosylated angucycline. I was also able to generate another new glycosylated derivative of angucycline through gene disruption of tailoring enzymes. In this research, I isolated two novel angucycline derivatives and gained new insights into the glycosylation steps in the biosynthesis of Sch47554 and Sch47555. Next, I engineered the regulatory elements in Streptomyces sp. SCC-2136 through the overexpression and targeted gene disruption approaches for enhanced production of pharmaceutically important angucyclines. The highest titer of Sch47554 was achieved in Streptomyces sp. SCC-2136/ΔschA4 (27.94 mg/L), which is significantly higher than the wild type. This work thus provides an initial understanding of functional roles of regulatory elements in the biosynthesis of Sch47554 and Sch47555 and several engineered strains with enhanced production of Sch47554. Last, I isolated a carotenoid-producing endophytic bacterium from the leaves of the yew tree and optimized the fermentation conditions for an improved yield of zeaxanthin diglucoside up to 206 ± 6 mg/L. With the introduction of an additional copy of the Pscrt gene cluster through an expression plasmid, the engineered strain Pseudomonas sp. 102515/pOKF192 produced zeaxanthin diglucoside at 380 ± 12 mg/L, which is 85% higher than the parent strain. This strain holds a great potential for the production of pharmaceutically important antioxidant agent, zeaxanthin diglucoside.

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.

This research focuses on the understanding and engineering of nonribosomal peptide biosynthetic pathways in Streptomyces coelicolor CH999, Escherichia coli BAP1 and Saccharomyces cerevisiae BJ5464-NpgA. The biosynthetic systems of indigoidine from bacteria and beauvericin/bassianolide from fungi were studied in this research. The production of these valuble products was significantly increased by enhancing their synthetic pathway with metabolic engineering approaches. Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. Indigo is a dark blue crystalline powder and has been known for more than 4,000 years. It is commonly used to dye cotton yarn for the production of denim cloth to make blue jeans but the chemical synthesis of indigo requires harsh conditions and use of a strong base. Indigoidine is a new natural blue dye that is vi assembled from two molecules of L-glutamine under the catalysis of indigoidine synthetase. We identified a novel indigoidine synthetic gene from the genome of Streptomyces chromofuscus ATCC 49982. The successful heterologous expression of Sc-indC in E. coli BAP1 give us a pretty good yield of indigoidine under the optimized conditions. The production of this blue dye was then further improved by introducing two additional genes, sc-indB and glnA, into the biosynthetic pathway. Beauvericins and bassianolide are anticancer natural products from fungi and are assembled by corresponding iterative nonribosomal peptide synthetases. The beauvericin (BbBEAS) and bassianolide (BbBSLS) synthetases were successfully reconstituted in S. cerevisiae BJ5464-NpgA, leading to the production of beauvericins and bassianolide, respectively. The production of beauvericins was significantly improved by co-expression of BbBEAS and ketoisovalerate reductase (KIVR). To better understand the synthetic strategy of fungal iterative NRPs, the module/domain of BbBSLS and BbBEAS were dissected and reconstituted in S. cerevisiae. The result shows the intermodular linker is essential for the reconstitution of the separate modules and the domain swapping results indicated the fungal iterative NRPSs use a liner biosynthetic route which is different than bacterial iterative NRPs. The in vitro reactions of C2 and C3 with monomer/dimer/trimerN-acetylcysteamines demonstrated that C2 forms the amide bond and C3 catalyses the synthesis of the ester bond. Beauvericin could be reconstituted in vitro through co-reaction of C2(BbBEAS) and C3(BbBEAS) with D-Hiv-SNAC and N-Me-L-Phe- SNAC. This work also provides an unprecedented tool for engineering fungal iterative NRPSs to yield ‘unnatural’ cyclooligomer depsipeptides with varied chain lengths.