| Modern bioprocessing applications require superior cellular traits, many stemming from unknown genes and gene interactions. Tolerance to toxic chemicals is such an industrially important, complex trait, which frequently limits the economic feasibility of the production of commodity chemicals including biofuel molecules. Chemical tolerance encompasses the improved ability of an organism to retain viability and/or grow under chemical stress.;We first examined the impact of overexpressing the autologous GroESL chaperone system on the tolerance of Escherichia coli to several toxic alcohols. GroESL overexpression enhanced cell growth and viability in all alcohols tested, including a 12-fold increase in total growth in 4% ethanol, a 2.8-fold increase under 0.75% n-butanol, a 3-fold increase under 1.25% 2-butanol, and a 4-fold increase under 20% 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in viable cell counts compared to a plasmid control strain under 6% ethanol, and a 3.5-fold and 9-fold increase under 1% n-butanol and i-butanol, respectively.;Then, using combinations of HSPs, we engineered a semi-synthetic stress response system capable of improving tolerance to high levels of toxic solvents. Co-overexpression of the HSPs on the plasmid pHSP produced a 200%, 390% and 78% increase in viable cell counts in 7% ethanol, 1% n-butanol or 25% 1,2,4-butanetriol, respectively.;Building upon the success of this semi-synthetic stress response system, we probed the genomic space of the solvent tolerant Lactobacillus plantarum to identify heterologous genetic determinants that impart complex solvent tolerance. Using two targeted enrichments, one for better viability and one for better growth under ethanol stress, we identified several beneficial and specialized heterologous DNA determinants that act synergistically with pHSP. In separate strains, a 209% improvement in survival and an 83% improvement in growth over previously engineered strains based on pHSP were generated. We then developed an even more complex composite phenotype of improved growth and survival by combining the identified L. plantarum genetic fragments generating a strain with simultaneous 3.7-fold better survival and 32% increased growth. This demonstrates the concept of a sequential, iterative assembly strategy for building multigenic traits by exploring the synergistic effects of genetic determinants from a much broader genomic space. |
