The specific goals of our laboratory are to establish an interdisciplinary approach to metabolic engineering, focusing on the fundamental physiology, biochemistry and molecular genetics of important organisms. In particular, we are studying key factors that regulate the synthesis of different biomolecules. We apply metabolic engineering in several different project areas. Among prokaryotic systems, we study amino acid metabolism in Corynebacterium glutamicum, bioremediation and bioconversion processes in Rhodococcus, and biopolymer synthesis among Gram-negative bacteria such as Ralstonia eutropha. Among eukaryotic systems, we are studying lipid biosynthesis and embryogensis in oil palm and the accumulation of secondary metabolites in tropical plants
Metabolic Engineering in Corynebacterium glutamicum: C. glutamicum is a nonpathogenic, gram-positive, food-grade microorganism with a long fermentation history, and thus is potentially useful as a host strain for producing a number of recombinant DNA products. We have developed fundamental genetic tools enabling one to directly address questions of gene organization, structure and regulation at the molecular level. The primary objectives of recent research include:
Using DNA microarrays and functional genomics to dissect the genetic elements responsible for amino acid production in Corynebacterium;
Studies on the role of central carbon metabolism in Corynebacterium glutamicum, particularly with respect to the metabolism of pyruvate and acetyl CoA;
Mechanisms by which regulation of enzyme activities affect the accumulation and secretion of amino acids such as lysine, methionine, threonine and isoleucine.
Bioconversion Processes: Bacteria in the genus Rhodococcus have been found to metabolize a number of recalcitrant environmental contaminants such as halogenated hydrocarbons, pesticides and aromatic compounds. This property makes Rhodococcus valuable in bioremediation strategies. Members of this genus have also proven useful in bioconversion processes in which simple substrates can be metabolized to more complex structures. Because of the stereoselectivity and stringent substrate specificity of the enzymes involved, microbial systems can, in principle, be used to synthesize molecules that would be difficult to synthesize chemically. Developing a fundamental understanding of the physiology and molecular biology of Rhodococcus is an important prerequisite for applying these organisms in industrial processes.
We have been studying several isolates of Rhodococcus including one that can convert the bicyclic hydrocarbon indene into indandiol, an important precursor to the HIV protease inhibitor Crixivan™, and others that can metabolize compounds such as toluene, aniline or biphenyls. We are examining the production of secondary metabolites in these organisms, including pigments and possible new antibiotics.
Biopolymer Engineering: While many microorganisms store carbon in the form of glycogen or other polysaccharides, others store carbon as natural polyesters (bioplastics). These polyesters, known collectively as polyhydroxyalkanoates (PHAs), have a range of valuable properties including biodegradability and thermoplasticity. These features make microbial polyesters interesting for the newly emerging field of biopolymer engineering, a major new application for recombinant DNA technology. Our research investigates the genetic and molecular basis for biopolymer synthesis, examining both how the cell regulates the accumulation and composition of the polymer as well as the physiological processes that support PHA biosynthesis.
The genetic loci for PHA biosynthesis from Ralstonia eutropha H16 and Chromatium vinosum have been characterized. Heterologous expression of these genes in different species has allowed us to characterize the biochemical pathway that controls the amount and type of polymer that accumulates. Biochemical studies of the polymerase enzyme, PHA synthase, have revealed novel structures in the active site of the enzyme, suggesting a unique reaction mechanism. By understanding the biochemistry and molecular biology of the biosynthetic pathways, we will be able to develop rational strategies for the controlled synthesis of PHA biopolymers.
Malaysia-MIT Biotechnology Partnership Program: Together with Professor Rha (MIT Biomaterials Science and Engineering Lab) and other labs at MIT, we have begun a collaboration with the Malaysian Palm Oil Board to apply the principles of metabolic engineering to modify fatty acid synthesis in the oil palm (Elaeis guineensis). We have been using DNA microarrays to study the regulation of gene expression in somatic embryos and also to identify genes with useful expression patterns in oil palm. Using a system of bioreactors and mathematical modeling, we are developing metabolic models of the behavior of oil palm cells in culture. This work supports a major initiative in Malaysia to bolster the production of oil palm-derived oils for food and other uses. Collaborations with the Forest Research Institute of Malaysia focus on the accumulation of natural compounds in native Malaysian plants that have been used in traditional medicines and may have pharmaceutical and nutraceutical value. Plants from the species Centella asiatica and Eurycoma longifolia have been implicated for treating a range of disorders. In collaboration with a network of Malaysian medical and academic institutes, we have been working to identify the chemical basis for these properties by isolating compounds from plant extracts and developing bioassays for specific activities. We are also examining the physiological responses mammalian cells have when exposed to extracts from these plants through a variety of techniques including gene expression analyses, cell viability assays, and atomic force microscopy. We have found that compounds present within these extracts influence gene expression in a number of important pathways, including hormone production, stress responses, nitric oxide metabolism and wound healing responses.
Choi, D.S., Andrade, M.H.C., Willis, L.B., Cho, C., Schoenheit J., Boccazzi P., Sambanthamurthi R., Sinskey A.J., and Rha C.K. Effect of Agitation and Aeration on Yield Optimization of Oil Palm Suspension Culture. J. Oil Palm Research Special Issue 1:23-34 (2008).
Rafat, M., Fong K.W., Goldsipe A., Stephenson B.C., Coradetti S.T., Sambandan T.G., Sinskey A.J., and Rha C.K. Association (micellization) and partitioning of aglycon triterpenoids. J. Colloid Interface Science. In Press (2008).
Abdullah, M.A., Rahmah A., Sinskey A.J., and Rha C.K.. Cell Engineering and Molecular Pharming for Biopharmaceuticals. Open Med. Chem. J. 2:49-61 (2008).
Willis, L.B.,. Lessard P.A, Parker J.A., O’Brien X.M., and Sinskey A.J. Functional annotation of oil palm genes using an automated bioinformatics approach. J. Oil Palm Research 1:35-43 (2008).
Willis, L.B., Gil G.A., Lee H.L.T., Choi D.S., Schoenheit J., Ram R.J., Rha C.K., and Sinskey A.J. Application of spectroscopic methods for the automation of oil palm culture. J. Oil Palm Research 1:1-13 (2008).
Thiers, F.A., Sinskey A.J., and Berndt E.R.. Trends in the globalization of clinical trials. Nat. Rev. Drug Discovery 7:13-14 (2008).
Kurosawa, K., Ghiviriga I., Sambandan T.G., Lessard P.A., Barbara J.E., Rha C.K., and Sinskey A.J. Rhodostreptomycins, Antibiotics Biosynthesized Following Horizontal Gene Transfer from Streptomyces padanus to Rhodococcus fascians. J. Am. Chem. Soc. 130(4):1126-7 (2008).