Comprehensive Molecular BioEngineering - Projects
Comprehensive Molecular BioEngineering

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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.

Microbial Consortia for Conversion of Mixed Sugars

Georgia and the southeastern U.S. are rich in natural biomass resources such as forest and agricultural residues. There has been a long interest in developing microbial processes to convert these complex residues into fuels and chemicals. The residues must first be broken down by thermal and/or chemical treatment (i.e., hydrolysis) into a complex and variable mixture. One of the technical challenges for a microbial process is that the organisms used to produce fuels and chemicals cannot utilize all the sugars in the complex mixture, nor can they handle some inhibitors that are generated during the hydrolysis process. Typical sugars found in lignocellulosic hydrolysate include glucose, xylose, arabinose, galactose and mannose. In our research, these challenges are address by engineering the microbial system in which the conversion takes place. The concept relies on using microbial strains which can only metabolize a single carbon source and then designing an efficient process using a consortium of these nutritionally selective strains. For example, we have developed a microbial strain which can consume xylose, but cannot consume arabinose and glucose. With this approach, we have developed a process first to remove the inhibitor acetic acid from lignocellulosic hydrolysate, and second to convert the remaining sugars into desirable products. Because the approach is general, it can be applied to the production of any microbial product. The technology constitutes a major advance in the conversion of lignocellulose into fuels and chemicals.

Production of Succinic Acid by Recombinant Escherichia coli

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.

Increased Yield of Recombinant Proteins by Diverting Carbon to Oxaloacetate

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.

Production of Pyruvic Acid and Alanine by Recombinant Escherichia coli

Pyruvic acid is a three carbon ketoacid synthesized at the end of glycolysis. Pyruvate is an important raw material for the production of L-tryptophan, L-tyrosine, 3,4-dihydroxyphenyl-L-alanine, and for the synthesis of many drugs and biochemicals. Alanine is the smallest chiral amino acid and can be synthesized in one step from pyruvate via the enzyme alanine dehydrogenase. We are studying the production of pyruvate, alanine and other compounds derived from pyruvate in Escherichia coli. In order to accumulate compounds which are so central in metabolism, the cells must be unable to metabolize pyruvate. We have therefore focussed our research on strains which have mutations in the pyruvate dehydrogenase complex, PEP carboxylase and other enzymes. In addition to accumulation of these compounds, we are studying the affect of these genetic perturbations on cell physiology. Currently, ideal operating conditions lead to the accumulation of about 90 g/L pyruvate in 40 hours, or 32 g/L alanine in 24 hours.