Jilai Zhou is a PhD candidate at Thayer School of Engineering.
As a substitute for liquid transport fuel, bioethanol has become a focus in industrial biotechnology. Unlike the first generation bioethanol derived from feedstock, cellulosic ethanol not only reduces the dependency of fossil fuel and the carbon dioxide emission, but also avoids the consumption of starch or sugars important for humans. An efficient way to convert cellulose to ethanol at low cost is via consolidated bioprocessing (CBP).
Clostridium thermocellum, a gram-positive bacterium, is a candidate for CBP because of its ability to rapidly solubilize cellulose. However, C. thermocellum has a low ethanol yield when it ferments cellulose, and the main byproducts during the fermentation process are organic acids such as lactic acid, acetic acid and amino acid. My research focuses on developing new strains of C. thermocellum via metabolic engineering, enabling C. thermocellum to produce ethanol as the only organic product in the fermentation process.
In the literature, several other microorganisms have been successfully developed to produce ethanol at high yield and titer through genetically modifying genes that are responsible for byproduct formation. To eliminate production of lactic and acetic acid, we deleted the ldh and pta genes, however the resulting carbon flux was diverted to additional amino acid production instead of ethanol production. In some strains, amino acids production, mainly L-valine and L-alanine, could occupy over 20 percent of the total carbon flux among the fermentation products.
Since amino acids are necessary for growth, they cannot be deleted. Amino acid production requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. In the central metabolism of C. thermocellum, there is a pathway known as “malate shunt”, which has a transhydrogenation activity, generating NADPH from reduced nicotinamide adenine dinucleotide (NADH). One of our strategies is to disrupt this pathway via target gene deletion. After several attempts, we successfully created this new mutant. Based on this mutant, we also created several other mutants with the combination of other gene deletions, such as pta gene.
However, analyzing amino acid productions of these strains requires special chemical derivitization and special analysis equipment. This analysis cannot be accomplished by the high performance liquid chromatography (HPLC) equipment in our lab at Dartmouth and have to be sent out for analysis.
With the support of Graduate Alumni Research Award, I am able to analyze the all the excreted amino acids produced by mutant strains. Compared to parent strain, the amino acid productions in the mutants were significantly reduced, but the carbon flux didn’t completely go to ethanol production either. This work provides insights into the amino acid formation pathway and regulation of central metabolism of C. thermocellum. It also guides us to investigate the intracellular redox balance and nitrogen metabolism of C. thermocellum, which are both highly related to the amino acid production. I believe it will help us develop a metabolic engineering strategy to further improve ethanol production.
