Russell B. Williams

Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination

The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.

Briarane Diterpenes Diminish COX-2 Expression in Human Colon Adenocarcinoma Cells

Exploration of a soft coral (Briareum sp.) from Vanuatu led to the isolation of three new briaranes, designated brialalepolides A (1), B (2), and C (3). Compounds 2 and 3 reduced the expression of COX-2 in human colon adenocarcinoma cells, as well as in murine macrophage cells. This is significant because the metabolic products of COX-2 have been implicated in the pathogenesis of colon cancer and other diseases.

Diterpenes from the Endangered Goldenrod Solidago shortii

Species extinction is tantamount to loss of chemical diversity, and so it is important to seize all opportunities to study species on the brink of extinction. Such studies are often hampered by the limited material available, but that obstacle is surmountable through collaboration with botanical gardens and advances in instrumentation. The goldenrod Solidago shortii is one example of an endangered species native to the United States. From S. shortii, one known diterpene (1), two new diterpenes (2 and 3), and three new hydrolysis products (4-6) are described. This work was made possible through collaboration with the Missouri Botanical Garden and with the use of highly sensitive microcryoprobe NMR technology for structure elucidation and VCD spectroscopy for the determination of absolute configuration.

Digging Deep for New Compounds from the Compass Plant, Silphium laciniatum

The compass plant, Silphium laciniatum, is an iconic perennial plant of the North American tallgrass prairie. The plants of the tallgrass prairie historically have been subjected to a number of biological and environmental stresses. Among the adaptations developed by S. laciniatum is a large deep taproot. An investigation of the secondary metabolites found in the root of a S. laciniatum specimen has led to the identification of 15 new terpenoids (3-8, 10-17, and 22), which were screened for cytotoxic activity in the NCI-H460 human large-cell lung carcinoma cell line.

Cytotoxic and antibacterial beilschmiedic acids from a gabonese species of Beilschmiedia

High-throughput natural products chemistry methods have facilitated the isolation of eight new and two known beilschmiedic acid derivatives from the leaves of a Gabonese species of Beilschmiedia. This class of compounds appears to be unique to two genera of the Lauraceae, and exhibits remarkable structural complexity for a fatty acid. Eight of the compounds were isolated in microgram quantities necessitating the use of a 1.7mm MicroCryoProbe to obtain NMR data, including a complete HMBC, for structure elucidation and dereplication. All of the compounds were screened for cytotoxic activity against NCI-H460 human lung cancer cells and antibacterial activity against a clinical isolate of methicillin-resistant Staphylococcus aureus. Results from enzymatic assays provided evidence that the observed activity was not the result of membrane disruption as one might expect from lipophilic compounds such as these.