Date of Award:

12-2018

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biological and Irrigation Engineering

Advisor/Chair:

Jixun Zhan

Co-Advisor/Chair:

Ronald Sims

Third Advisor:

David W. Britt

Abstract

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.

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