Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress

ABSTRACT Synthetic acid tolerance, especially during active cell growth, is a desirable phenotype for many biotechnological applications. Natively, acid resistance in Escherichia coli is largely a stationary-phase phenotype attributable to mechanisms mostly under the control of the stationary-phase sigma factor RpoS. We show that simultaneous overexpression of noncoding small RNAs (sRNAs), DsrA, RprA and ArcZ, which are translational RpoS activators, increased acid tolerance (based on a low-pH survival assay) supra-additively up to 8500-fold during active cell growth, and provided protection against carboxylic acid and oxidative stress. Overexpression of rpoS without its regulatory 5 0 -UTR resulted in inferior acid tolerance. The supra-additive effect of overexpressing the three sRNAs results from the impact their expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks, thus leading to superior tolerance during active growth. Unlike the overexpression of proteins, overexpression of sRNAs imposes hardly any metabolic burden on cells, and constitutes a more effective strain engineering strategy

Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes

In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA

Exploring the Heterologous Genomic Space for Building, Stepwise, Complex, Multicomponent Tolerance to Toxic Chemicals

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 <i>Lactobacillus plantarum</i> 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 <i>L. plantarum</i> 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 <i>n</i>-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