There is an increasing demand for the use of the biodegradable and renewable carbon source starch in a variety of industries. The rate-limiting enzyme in the bacterial glycogen and plant starch biosynthesis pathways is ADPGlucose Pyrophosphorylase (ADPG PPase, glgC gene product) which is regulated by the binding of various allosteric effector molecules depending on the carbon utilization pathway of the organism. A complete molecular comparison of this enzyme family will allow us to perform rational engineering. The successful engineering of ADPG PPase would allow for the overproduction of starch in transgenic plants. Further, increased starch synthesis in transgenic plants could increase photosynthesis (and biomass) by decreasing feedback inhibition by organic phosphates. This project is focused on kinetic, physical, and molecular studies of the uniquely regulated bacterial ADPG PPases from a variety of sources including Rhodobacter sphaeroides, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palustris, Deinococcus radiodurans, and Chlamydia trachomatis. The specific aims of this research project include I: Cloning and expression of novel glgC genes and characterization of the recombinant enzymes; and II: Protein engineering of the bacterial ADPG PPases utilizing the techniques of site-directed, truncation, chimera, and random mutagenesis well as DNA shuffling. The comprehensive approach in this project will be guided by alignment studies, a recently solved structure, molecular modeling and in silico ligand docking. Characterization of the recombinant ADPG PPases will include the measurement of binding of ligands using affinity capillary electrophoresis to complement steady-state kinetic assay data. A longer term goal includes transforming genes coding for highly active bacterial ADPG PPases into Arabidopsis thaliana leaves to determine the effects on starch accumulation, photosynthesis, and biomass.
This project is well suited to training students at a primarily undergraduate institution in the theory and practice of biochemistry, analytical chemistry, molecular biology, bioinformatics, and molecular modeling within the framework of a larger interdisciplinary biotechnology project. Some of the research experiments and findings will be incorporated into lecture and lab class activities. Participating students represent three laboratories at two different California State University campuses as well as summer research students from both high schools and local community colleges. The background and experience that is gained by students in this project will open attractive opportunities in both academic and industrial careers.
Our research has been focused on the complete molecular characterization of the glycogen and starch biosynthesis pathways in bacteria and plants using the tools of modern biochemistry, molecular biology, and biotechnology. The highly regulated ADPGlucose Pyrophosphorylase (ADPG PPase) serves as the rate-limiting enzyme in these pathways. There is increasing demand for the renewable and biodegradable carbon source starch in the food, chemical, electronic, and pharmaceutical industries. Starches and modified starches serve as starting materials for the synthesis of bio-ethanol and organic acids and in the making of biodegradable plastics, packaging materials, adhesives, and detergents, lessening dependence on oil resources. These renewable and biodegradable carbon sources can serve as inexpensive starting materials for bio-ethanol, organic acids, and antibiotic synthesis and have great potential for use in the making of specialty plastics, adhesives, detergents, surfactants, and packaging materials. The activity of ADPG PPase is modulated by the binding of various metabolites that serve as allosteric effector molecules (which activate or inhibit the enzyme) depending on the carbon utilization pathway of the organism. A complete molecular comparison of this family of diverse enzymes will allow us to perform protein engineering and directed evolution with the goal of enhancing function. The successful engineering of ADPG PPase as well as other enzymes in the pathway would allow for the overproduction of novel starches in transgenic plants. Given that enhanced starch biosynthesis has been recently shown to stimulate photosynthesis, engineered plants and/or microbes may be able to more efficiently sequester carbon thus removing CO2 from the atmosphere to combat climate change. Our eclectic approach has led to the forging of several important interdisciplinary collaborations to complement our biochemistry expertise with x-ray crystallography and molecular modeling, analytical chemistry, and plant biotechnology. Seminal data we have produced include the first cloned and expressed ADPG PPase genes from particular regulatory classes of bacteria, the first recombinant purification and characterization of several of these enzymes (some of them comprised of two subunits), the identification of functional amino acids by mutagenesis (in both the active and regulatory sites), and the first three-dimensional structure of a bacterial ADPG PPase—an achievement that had eluded researchers for decades. The availability of high quality structures has enabled us to incorporate molecular modelling which has been an extremely useful tool for generating additional hypotheses about structure-function relationships. We have also characterized three different thermophilic ADPG PPases. This puts us in a very good position to begin a systematic investigation of the mechanism of heat stability, an attractive trait for industry and some crop plants. With a number of engineered enzymes in hand, we are well poised (via collaboration) to express some of our engineered proteins in both transgenic model and crop plants. Engineering this important pathway will meaningfully contribute to agricultural and environmental biotechnology. This project has been well suited to training diverse students in the theory and practice of biochemistry, analytical chemistry, molecular biology, bioinformatics, and molecular modeling within the framework of a larger interdisciplinary biotechnology project. Of equal importance to the research accomplishments has been the role of student researchers. Averages of 15-20 students per semester are involved in research projects in my laboratory, including many community college students as well as high school students during the summer. The technical, critical thinking, and communication skills gained have made these students attractive candidates for competitive graduate schools and industrial positions. These students have successfully gone on to many high quality Ph.D. programs including UC Riverside, UC Irvine, USC, UCLA, Yale, Johns Hopkins University, Vanderbilt, and the University of Wisconsin. In addition, many of the Meyer Lab students have gone on to top health professions schools (including at UCLA, UC San Diego, Harvard, and UC San Francisco) and industry positions (including Amgen, Allergan, BioRad, and Invitrogen). At the undergraduate and MS level, these students have won many awards including several Cal State system-wide and numerous regional and national poster competitions and travel awards. This research has also been successfully integrated into the curriculum of an advanced lab course (CHEM 472B) in which students generated new mutations and contributed to the purification and initial characterization of a number of altered proteins. The PI and research students have also actively participated in outreach activities, science demonstrations, and seminars to junior high and high school audiences as well as senior citizens.
