Claire Vieille Research Interests
Research in my lab focuses on biotechnology, more particularly on the design and development of industrial biocatalysts. Among the many industrial applications of bioprocesses are the use of microbial and enzyme biocatalysts in biochemical and biomaterial production, drug manufacture, sensors and diagnostics, and food and feed production. Our research on enzyme biocatalysts focuses primarily on enzymes from thermophiles and hyperthermophiles. With their high thermostability and solvent tolerance, these enzymes are attractive candidates for industrial processes, which typically require catalysts that are robust, highly active, and have high chemical yields. These enzymes are also ideal model proteins to study the mechanisms underlying protein thermostability. Our work on microbial catalysts focuses on organic acid (succinate and aspartate) production by a natural high-succinate producing bacterial species.
Enzyme engineering. Our work on enzymes from thermophiles and hyperthermophiles focuses on six enzymes: 1) adenylate kinase, a small protein used to study how flexibility relates to stability and activity; 2) xylose (glucose) isomerase used in high fructose corn syrup manufacture; 3) secondary alcohol dehydrogenase (SADH) used in chiral chemical synthesis;
4) alkaline phosphatase used in diagnostics; 5) aldose reductase, to be used to produce sorbitol from glucose; and 6) mannitol dehydrogenase to be used in combination with xylose isomerase for mannitol production from glucose. With the exception of aldose reductase (originating from a mesophilic yeast), these enzymes originate from the thermophile Thermoanaerobacter ethanolicus (SADH) and from the hyperthermophiles Thermotoga maritima and Thermotoga neapolitana, and they are optimally active at temperatures above 80°C. The goals of our research are to understand the molecular determinants for enzyme thermostability and activity; and to redesign the enzymes for enhanced activity, new substrate specificity, and stability.
Enzyme stability properties are studied by differential scanning calorimetry and kinetic inactivation studies (most enzymes), as well as by NMR relaxation and hydrogen-deuterium exachange studies (adenylate kinase). Stability properties are related to sequence and structural information, in collaboration with protein crystallographers. Engineering strategies (structure-based site-directed mutagenesis or directed evolution) are designed based on the engineering objective and structural information availability. Using a combination of structure-based site-directed mutagenesis and directed evolution, we engineered a xylose isomerase mutant that is five times more catalytically efficient at 60°C (pH 5.5) than the wild-type enzyme is at 80°C (pH 7.0). While wild-type SADH does not use benzylacetone as substrate, the structure-base designed W110A mutant now uses benzylacetone with high efficiency, and produces (S)-4-phenyl-2-butanol in over 99% enantiomeric excess.
Oxidoreductases as industrial catalysts. The fact that enzymatic oxidoreduction reactions consume stoichiometric amounts of expensive cofactors and the absence of convenient and robust cofactor recycling method have long hindered the use of dehydrogenases in industrial processes. In collaboration with Drs. Mark Worden and Scott Calabrese-Barton (MSU Dept. of Chemical Engineering and Material Science) who are developing the bioelectrochemical reactors, we are exploring electricity-based cofactor recycling in enzymatic oxidoreductions. Bioelectrochemical reactors are used to explore direct glucose conversion to mannitol using thermostable xylose isomerase and mannitol dehydrogenase co-immobilized on the working electrode. Because SADH’s ketone and alcohol substrates are often poorly soluble in aqueous solutions, the behavior of bioelectrochemical reactors is also tested with SADH in the presence of organic solvents.
Succinate production by Actinobacillus succinogenes . If the cost of bio-based succinate was competitive with that of the petrochemical-based alternative, maleic anhydride, a US $15 billion market could be based on succinate for producing bulk chemicals such as 1,4-butanediol (a precursor to “stronger-than-steel” plastics), diethyl succinate (a green solvent for replacement of methylene chloride), and adipic acid (nylon precursor). The development of a cost-effective industrial succinate fermentation will rely on organisms able to produce high concentrations of succinate and at high rates. Isolated from a cow rumen, the Gram negative bacterium, A. succinogenes, is the best natural succinate producer known (producing up to 80 or 100 g/l succinate from glucose), but it also produces acetate, formate, and ethanol as byproducts. Our goal is to engineer A. succinogenes to efficiently produce succinate as the sole fermentation product. Previous studies have involved 1) developing an A. succinogenes-Escherichia coli shuttle plasmid vector; 2) identifying the main active intermediary metabolic pathways using in vitro enzyme assays; 3) developing a chemically defined growth medium for A. succinogenes. This medium is used in 4) 13C-labeling experiments to confirm active metabolic pathways and measure intracellular metabolic fluxes, in an approach called metabolic flux analysis (MFA); and 5) identifying genes that can be used as markers in constructing knockout mutants.
Our MFA studies show that during growth on glucose, NADPH is produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Significant forward and reverse fluxes through malic enzyme and/or oxaloacetate decarboxylase indicate that phosphoenolpyruvate, oxaloacetate, malate, and pyruvate represent four nodes for flux distribution between succinate and alternative fermentation products. High NaHCO 3 and H 2 concentrations increase succinate production at the expense of acetate and formate production. They do so by decreasing the amount of flux shunted by through malic enzyme and/or oxaloacetate decarboxylase from the succinate pathway to the formate, acetate, and ethanol pathway, and by increasing the pyruvate carboxylating flux. Pyruvate and formate dehydrogenase fluxes vary in response to the different redox demands imposed by the different NaHCO 3 and H 2 concentrations. Overall, these metabolic flux changes allow A. succinogenes to maintain a constant growth rate and biomass yield in all conditions. These metabolic constraints should be taken into consideration for designing effective metabolic engineering strategies to increase A. succinogenes succinate production.
Current and future work involves 1) evolving mutant strains that grow optimally on the most abundant lignocellulose sugars (e.g., xylose and L-arabinose), and on glycerol, a byproduct of biodiesel production; 2) characterizing the metabolism of these new strains by MFA; 3) developing a knockout methodology for A. succinogenes; and 4) engineering A. succinogenes for succinate homofermentation.