Tetracyclines have been prescribed as broad-spectrum antibiotics and have recently been used in anticancer therapies. The objective of this research is to understand the metabolic pathway, the molecular assembly, and the combinatorial potential of tetracycline biosynthesis. Four specific aims will be pursued: (1) Reconstitute and understand the assembly of the amidated tetracycline backbone; (2) Reconstitute the first tetracyclic intermediate in oxytetracycline (oxy) pathway and use metabolic engineering to produce a novel C8-hydroxylated analog of the intermediate; (3) Reconstitute the tailoring steps in oxy pathway and construct a series of strains that can produce valuable metabolic intermediates; (4) Engineered biosynthesis of novel glycosylated analogs of tetracycline through shuffling of heterologous secondary metabolic pathways. The research and education efforts will be integrated through the following mechanisms: 1) Two recently developed, core biotechnology courses will be enriched to increase critical thinking and active learning by the students; 2) Community outreach will be emphasized by inviting underprivileged minority students in the greater Los Angeles metropolitan area to learn research during the summer months, as well as participating in university-community events to promote science education and life-long learning.
The goal of this work is to uncover the ways nature synthesize improtant antibiotics. With this knowledge, we can engineer microorganisms to produce better drug molecules. In addition, the work can also provide fundamental insights into the different chemical and enzymatic tools nature uses to assemble complex molecules. Intellectual Merit: We targeted the tetracycline biosynthetic pathway. Tetracycline belongs to the polyketide family of compounds and is a very important, front-line antibiotics. In the last five years, we completely mapped out how tetracycline is produced from the streptomyces organism. We studied in detail, the initiation, elongation, modification and termination of the biosynthetic pathways. We identified numerous key enzyme that control which buiding block to select for the assembly. We also uncovered several novel enzymes that were previously unknown. In total, this work generated ~20 manuscripts and signficantly added to our knowledge of tetracycline and polyketide biosynthesis. Broader Impact: Scientifically, our studies on tetracycline fills an important knowledge gap in our understanding of microbial chemistry. This project also trained several new graduate students and postdocs. Two trainees became faculty members at leading chemical/biological engineering departments. In addition, we hosted summer research high students in our lab during the award period. These students were exposed to university research environment, as well as learning laboratory research skills that can prepare them for a career in science and engineering.
