Sunday, April 29, 2007

Directed strain selection: Strategies for engineering productivity in E. coli

Tanya Warnecke, Michael D Lynch, Nicholas Sandoval, and Ryan T. Gill. Department of Chemical and Biological Engineering, University of Colorado, 1111 Engineering Drive, UCB 424, Boulder, CO 80309

Selection is powerful yet poorly understood, which limits strain-engineering efforts in a broad range of industrial biotechnology applications. We have developed a new genomic tool to address these issues.  SCalar Analysis of Library Enrichments (SCALEs) is capable of assessing greater than 106 enrichment patterns corresponding to individual clones contained within comprehensive genomic libraries. That is, SCALEs allows one to track the relative concentration of each clone that encodes specific genetic information to assess enrichment and dilution patterns throughout a broad range of selections. We have used this approach to improve understanding of how i) different genotypes are capable of conferring the same phenotype, ii) different phenotypes contribute to overall fitness, iii) genetic adaptations that are beneficial in one environment can be costly in another, and iv) selection strategy can dictate enrichments directed at the same phenotype.  We have demonstrated this within the context of engineering improved organic acid (3-hydroxypropionic acid (3HP)) production in E. coli. In this case a general selection indicated that increased copy of carbon catabolism, transporters, or biofilm mediation genes were the primary means for increasing fitness in a continuous flow reactor. We then redesigned our selections to enrich for transporter functions, which may work to decrease intracellular accumulation of 3HP, as opposed to the less desirable catabolic or biofilm functions. Finally, we used this information to identify specific metabolic compounds that when added to the growth media are capable to improving strain fitness in the presence of normally inhibitory levels of 3HP.