Stress, Solvent Production & Tolerance
(in Clostridium acetobutylicum) 

E. Terry Papoutsakis, Chris Tomas, Keith Alsaker, Hendrik Bonarius, He Yang, Jeff Beamish

Department of Chemical Engineering,

 & Neil Welker (BMBCB)

 Northwestern University, Evanston, IL

 

Understanding solvent (and other toxic chemical) tolerance of microorganisms is crucial for the production of chemicals, bioremediation, and whole-cell biocatalysis. Past efforts to produce tolerant strains have relied on selection under applied pressure and chemical mutagenesis, with some good results, but not consistently so.  We desire to examine if Metabolic Engineering (ME) and genomic approaches can be used to construct more tolerant strains for bioprocessing. The accepted dogma is that toxicity is due to the chaotropic effects of solvents on the cell membrane.   We have found that in C. acetobutylicum, several well-defined genetic modifications not related to membrane function impart solvent tolerance (by 40-70%) without strain selection. This suggests that we need to re-examine the accepted dogma. The objective of this research is to identify genes that contribute to solvent tolerance and to use genetic modifications (involving these genes) to generate solvent tolerant strains. A potential mechanism to overcome solvent toxicity is through the over-expression of heat shock proteins, possibly providing increased protein stability.   A C. acetobutylicum strain, 824(pGROE1), over-expressing the molecular chaperone genes groES and groEL, under control of the clostridial thiolase promoter, was created to examine this hypothesis.  Final acetone and butanol titers in the over-expressing strain were 66% and 56% higher than in the respective control strain, 824(pSOS95del).  Both 824(pGROE1) and 824(pSOS95del) exhibited a sustained solvent production profile (120 hours versus 40 hours for wild type) with increased acetone and butanol formation fluxes.  Western analysis of 824(pGROE1) confirmed over-expression of GroEL (3-180 fold) and revealed increased levels of proteins involved in solvent formation.  DNA-microarrays suggest that the presence of a control plasmid in C. acetobutylicum results in a generalized stress response with decreased cell motility and chemotaxis. DNA-array analysis of various stresses points to changes to several important cellular programs and can serve as a roadmap for choosing other genes for possible ME intervention.

Acknowledgements: Supported by Grants by the National Science Foundation (BES-9911231) & EPA (R-82856201-0)

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