The Center for Molecular BioEngineering (CMBE) focuses on research for the improvement of biological processes through metabolic engineering. Metabolic Engineering involves targeted alteration of biochemical pathways toward the goals of increased yield and productivity of biological products, or for enhanced biodegradation capabilities. Other general objectives of CMBE are to add economic value to underutilized food, agricultural, textile, and paper resources in the state of Georgia through Bioprocessing. CMBE has facilities for the maintenance of both anaerobic and aerobic microorganisms, and molecular biology techniques are routinely practiced on procaryotes and eukaryotes. We also conduct DNA microarray experiments to elucidate gene expression in relation to phenotype and fermentation performance.

• Bioactive Peptides
• Succinic Acid
• Recombinant Proteins
• Pyruvic Acid and Alanine

Several other current projects cannot be described because of intellectual property concerns or because of confidentiality agreements made with industries.

Bioactive peptides are small peptides (about 20 amino acids on average) that elicit a wide range of biological activity. Since many of these peptides function by binding to and inhibiting target proteins with incredible specificities, they have been used for several years as therapeutic agents in medicine. Researchers have also been trying to derive synthetic bioactive peptides for antibacterial, anticancer and antiviral applications by using combinatorial peptide systems and in vitro chemical approaches. Although there is enormous potential for the development of synthetic inhibitor peptides using this technology, inhibitor peptides synthesized in this way have problems of instability and degradation by peptidases in the host cells. Through recombinant DNA techniques, our laboratory has devised an intracellular approach which allows for the direct screening of 5 to 20 amino acid inhibitory peptides that can prevent the growth of Escherichia coli bacteria. Subsequent analysis has revealed that most of these inhibitory peptides possess specific motifs which presumably allow them to form stable active structures. We have found that the deliberate incorporation of these motifs into inhibitor peptides increases both the frequency at which the peptides can be created, and the potency of the resulting peptides. Because peptidases in prokaryotes and eukaryotes function similarly, the approach for stabilizing peptides in bacteria can be directly applied to develop human drugs. We are applying this technology to 1) trivially stabilize current peptide drugs by incorporating the discovered motifs, and 2) apply our intracellular approach to isolate new peptide drugs.

Succinic acid is a four carbon dicarboxylic acid which has diverse applications in the food, pharmaceutical and cosmetics industries, and can also serve as a four carbon building block for polymers. As a tricarboxylic acid cycle intermediate, succinic acid is a ubiquitious biochemical which cells generally do not accumulate. However, during anaerobic growth, many organisms including Escherichia coli can accumulate small quantities of succinic acid. Our laboratory is working with a strain of E. coli (AFP111) carrying mutations in the pfl and ldh genes, and which therefore produces succinic acid as the major product of fermentation, but still produces substantial acetic acid. By transforming this strain with the pyc gene encoding for the enzyme pyruvate carboxylase, nearly complete conversion of glucose to succinic acid is accomplished, providing close to the maximum theoretical yield of 112% (mass succinic acid produced/mass glucose consumed). Our laboratory is developing means to optimize the productivity of succinic acid by modification of operational parameters toward eventual commercialization.

Recombinant proteins are a $10 billion industry, and include diverse products including immunoassay proteins, industrial enzymes and therapeutic proteins. When a protein is synthesized by a microorganism, production of the required amino acids places demands on carbon compounds from the glycolytic pathway and the tricarboxylic acid cycle. For example, ten of the twenty amino acids are derived from intermediates of the tricarboxylic acid cycle. In order for the tricarboxylic acid cycle to continue to operate, the compounds withdrawn for amino acid synthesis must be replenished, by have so called anaplerotic enzyme reactions. Indeed, at high growth rates and high rates of protein production, these anaplerotic reactions limit the amount of amino acids and hence proteins that can be synthesized. We have developed a technology to use the anaplerotic enzyme pyruvate carboxylase to divert carbon to the tricarboxylic acid cycle, and in so doing provide more carbon to protein synthesis. We have observed 50-70% increased protein yield using two model proteins, beta-galactosidase and catechol 2,3-oxygenase, in defined media. We are currently studying this technology for industrial clients.