title: Exploring the heterologous genomic space for building, stepwise, complex, multicomponent tolerance to toxic chemicals.
abstract: Modern bioprocessing depends on 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 producing commodity chemicals and biofuels. Chemical tolerance encompasses both improved cell viability and growth under chemical stress. Building upon the success of our recently reported semisynthetic stress response system expressed off plasmid pHSP (Heat Shock Protein), we probed the genomic space of the solvent tolerant Lactobacillus plantarum to identify genetic determinants that impart solvent tolerance in combination with pHSP. Using two targeted enrichments, one for superior viability and one for better growth under ethanol stress, we identified several beneficial 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 thus generated. We then developed a composite phenotype of improved growth and survival by combining the identified L. plantarum genetic fragments. This demonstrates the concept for a sequential, iterative assembly strategy for building multigenic traits by exploring the synergistic effects of genetic determinants from a much broader genomic space. The best performing strain produced a 3.7-fold improved survival under 8% ethanol stress, as well as a 32% increase in growth under 4% ethanol. This strain also shows significantly improved tolerance to n-butanol. Improved solvent production is rarely examined in tolerance engineering studies. Here, we show that our system significantly improves ethanol productivity in a Melle-Boinot-like fermentation process.
authors.affiliation: Molecular Biotechnology Laboratory, Dept. of Chemical & Biomolecular Engineering, the Delaware Biotechnology Institute, University of Delaware , Newark, Delaware 19711, United States.
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Zingaro
authors.forename: Sergios A
authors.initials: SA
authors.lastname: Nicolaou
authors.forename: Yongbo
authors.initials: Y
authors.lastname: Yuan
authors.forename: Eleftherios Terry
authors.initials: ET
authors.lastname: Papoutsakis
mesh_terms: D056804:Biofuels; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism / Q000633:toxicity; D015723:Gene Library; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D048191:Lactiplantibacillus plantarum / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D010957:Plasmids / Q000378:metabolism
chemical_list: D056804:Biofuels; D006360:Heat-Shock Proteins; D000431:Ethanol