Metabolic engineering of Escherichia coli for the efficient utilization of plant sugar mixture

Choudhary, Madhuresh
Major Professor
Jacqueline V. Shanks
Julie A. Dickerson
Monica H. Lamm
Committee Member
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Chemical and Biological Engineering

In recent years, metabolic flux analysis (MFA) has become an important tool in metabolic engineering. The MFA involves the quantification of intracellular metabolic fluxes in a microorganism. The result of MFA is a metabolic flux map that allows the systematic study of cellular responses to genetic and environmental perturbations. Carbon isotope labeling based MFA, with analysis by NMR or GC/MS, have gained wide use in the metabolic engineering community for estimating metabolic fluxes in central carbon metabolism. We are using MFA as a tool to engineer Escherichia coli sugar utilization regulatory (SURS) for the efficient consumption of sugar mixture.;E. coli SURS controls the utilization of different sugars as well as many other cellular functions. The SURS consist of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS) and by several global regulators including CRP (cAMP receptor protein), Mlc (controlling several glycolytic, gluconeogenic and glucose-related genes), and Cra (catabolite repressor/activator protein). The tight control of sugar utilization in E. coli by SURS results in the sequential consumption of other sugars in the presence of glucose, which results in low yields and productivities of the desired product.;We have constructed E. coli strain devoid of gene encoding ptsG. The ptsG is involved in the transport of glucose and plays an important role in carbon catabolite repression. The ptsG-strain didn't show diauxic growth and consumed glucose and xylose simultaneously. It grew slower than wild type grown on glucose and had a long lag time. Our flux analysis revealed that slow growth of ptsG mutant is due less efficient transport of xylose and glucose in the mutant which results in less available ATP for biomass synthesis.