(591c) Genomic Approaches to Generating Product-Tolerant Clostridium Acetobutylicum Strains for Bioprocess Applications | AIChE

(591c) Genomic Approaches to Generating Product-Tolerant Clostridium Acetobutylicum Strains for Bioprocess Applications

Authors 

Borden, J. R. - Presenter, Northwestern University
Papoutsakis, E. T. - Presenter, Northwestern University

Clostridium acetobutylicum ATCC 824 is important for the production of the solvents butanol and acetone, and fermentation-based production remained profitable well into the 1950s. However, end-product inhibition limits final product titers and therefore economic competitiveness as compared to petrochemical-derived solvents. In an effort to generate tolerant phenotypes and potentially solvent over-producing strains, we have investigated tolerance to organic acids and solvents by C. acetobutylicum ATCC 824 using DNA microarrays. To this end, DNA microarrays were used for transcriptional analysis of the metabolite stress response and also to evaluate the population dynamics of genomic libraries grown under metabolite stress.

We initially ascertained the short-term transcriptional response of C. acetobutylicum to physiologically relevant levels of the metabolites butyrate and butanol, providing a genome-wide view of differential gene regulation in response to individual metabolite stresses. Genes identified in these experiments as constitutively upregulated, including heat shock genes, osmolyte transporters, and components of the cell wall may play a key role in a tolerant phenotype. Therefore individual clones of a select set of these candidate genes were subsequently generated and characterized in order to ascertain each gene's specific ability to contribute to a phenotype exhibiting improved tolerance.

In parallel, genomic libraries were constructed from C. acetobutylicum genomic DNA, randomly sheared by sonication, ligated into suitable shuttle vectors, and electroporated back into 824. When these library-bearing cultures were subjected to evolutionary screening against incrementally increasing levels of bytyrate and butanol, distinct subsets of genes became clearly enriched from the original library population, as visualized by hybridization of plasmid library DNA to microarrays. Library-bearing C. acetobutylicum cultures were able to grow in media containing up to 205 mM (1.8% vol/vol) butanol or 110 mM (1%) butyrate, 25% higher than the peak levels found in a typical fermentation and over 100% higher than the tolerance of a plasmid control strain (100mM/0.9% butanol and 45mM/0.4% butyrate). Individual colonies, containing a single evolutionarily selected library insert, were retrieved directly from challenged cultures and evaluated for organic acid production and tolerance characteristics.

The utilization of multiple genomic approaches for phenotype evaluation provides several complementary advantages. First, the breadth of information from screening of genomic libraries is entirely dependent on library coverage, while transcriptional analysis of the stress response covers the entirety of the arrayed genome. In contrast, the screening of plasmid libraries allows for immediate characterization of selected library inserts, reducing the time for overall phenotype development. Therefore, multiple genome-scale methods can be utilized to complement each other and improve the overall breadth and speed of obtaining genetic information as well as the generation of relevant phenotypes.