Metabolic Engineering of Solvent Tolerance in Anaerobic BacteriaEPA Grant Number: R828562
Title: Metabolic Engineering of Solvent Tolerance in Anaerobic Bacteria
Investigators: Papoutsakis, E. T. , Welker, N. E.
Institution: Northwestern University
EPA Project Officer: Richards, April
Project Period: June 1, 2000 through May 31, 2003
Project Amount: $180,000
RFA: Technology for a Sustainable Environment (1999) RFA Text | Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development
Description:The objectives of Metabolic Engineering are to change the properties of a cell in order to achieve desirable cellular traits for bioprocessing. In the last few years, much effort has been devoted to understanding quantitative and genetic aspects of flux control in branched or linear metabolic pathways. In addition to maximizing the flux for a desirable product, the robustness and prolonged productivity of the biocatalyst (the cells) under realistic bioprocessing conditions is an equally important issue. In this sense, the ability of the cells to withstand "stressful" bioprocessing conditions (such as accumulation of toxic products, byproducts or substrates) without loss of productivity is a most significant goal. The difficulty - but also the intellectual and biotechnological challenge - is that the desirable phenotypic trait is determined by a sequence of genes or a complex regulatory circuit. The proposed research is precisely in this spirit: to understand and exploit the molecular basis which determines tolerance of bacterial cells (and in particular of the industrially important anaerobic clostridia) to solvents. This is a problem of both specific and general significance since it is encountered in the production of commodity chemicals from renewable resources, but also in bioremediation technologies, and the use of cells in biocatalysis involving toxic organic molecules.
Approach:Our hypothesis is that the molecular basis of what makes bacterial cells able to withstand high solvent concentrations can be used to metabolically engineer cells so that they can tolerate higher concentrations of solvents and related chemicals. This will make it possible to achieve much higher product (solvent) concentrations and cell densities in bioreactors and thus achieve productivities which are necessary to economically carry out the desirable bioprocess. As a model system we will use the anaerobe Clostridium acetobutylicum ATCC 824 (whose genome sequence was completed under the DOE's Microbial Genome Project). Using preliminary data, we argue that overexpression of heat-shock or stress proteins (HSPs) - which are molecular chaperones assisting protein folding and re-folding - confers increased tolerance to butanol, possibly due to the stabilizing effect of HSPs on solvent-producing enzymes. Based on information from other organisms, it is likely that specific solvent-resistance genes may be also involved in solvent tolerance. The function and effect of such genes will be also explored in order to generate strains with increased solvent tolerance. Specifically, we will test two hypotheses. The proposed research will be jointly funded by the National Science Foundation (NSF) and the US Environmental Protection Agency (EPA).
Hypothesis 1 (This portion of the research will be funded by the NSF): That increased butanol tolerance and the ability to produce butanol to high titers is enhanced by increased protein stability as a result of overexpression of HSPs of the groE and dnaK operons. First we will examine the effects of increased levels of the two native HSP families (DnaKJ, GroESL), both in combination and separately, on the ability of the recombinant strains to achieve higher butanol titers, and/or tolerate high butanol concentrations. We will also test the effects of increased levels of these HSPs on the in vivo stability of the AAD, CoAT and AADC enzymes (which catalyze the final steps of butanol an acetone formation). Second, will examine the effects of down-regulation of the expression of the dnaK operon genes to test if their absence reduces butanol titers and/or tolerance. The expression of these HSPs will be down regulated using either the gene-knockout or antisense RNA technologies.
Hypothesis 2 (This portion of the research will be funded by EPA): That butanol tolerance is also enhanced by the expression of native solvent/drug resistance genes identified on the basis of homology to genes from Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa. We will use Northern analysis to examine the patterns of expression of these genes in ATCC 824 grown with and without exogenous addition of butanol. If gene expression patterns show that any of these genes is possibly related to solvent tolerance, we will clone and either overexpress (or down regulate the expression of) this gene to verify its significance. Further studies will be pursued accordingly to understand the function of such genes and their protein products. Promising recombinant strains will be characterized in fermentation studies using flux analysis. In addition, the exploration of the industrial potential of new strains will be pursued. The proposed research provides unique opportun