Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.

A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors.

Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production.

A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination.

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.

Dissecting the assays to assess microbial tolerance to toxic chemicals in bioprocessing.

Microbial strains are increasingly used for the industrial production of chemicals and biofuels, but the toxicity of components in the feedstock and product streams limits process outputs. Selected or engineered microbes that thrive in the presence of toxic chemicals can be assessed using tolerance assays. Such assays must reasonably represent the conditions the cells will experience during the intended process and measure the appropriate physiological trait for the desired application.

Overexpression of fetA (ybbL) and fetB (ybbM), Encoding an Iron Exporter, Enhances Resistance to Oxidative Stress in Escherichia coli.

Reactive oxygen species are generated by redox reactions and the Fenton reaction of H2O2 and iron that generates the hydroxyl radical that causes severe DNA, protein, and lipid damage. We screened Escherichia coli genomic libraries to identify a fragment, containing cueR, ybbJ, qmcA, ybbL, and ybbM, which enhanced resistance to H2O2 stress. We report that the ΔybbL and ΔybbM strains are more susceptible to H2O2 stress than the parent strain and that ybbL and ybbM overexpression overcomes H2O2 sensitivity.

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

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.

Exploring the combinatorial genomic space in Escherichia coli for ethanol tolerance.

Strain tolerance to toxic chemicals is desirable for biologically producing biofuels and chemicals. Standard genomic libraries can be screened to identify genes imparting tolerance, but cannot capture interactions among proximal or distant loci. In search of ethanol tolerance determinants, we expanded the genomic space combinatorially by screening coexisting genomic libraries (CoGeLs) of fosmids (large inserts) and plasmids (smaller inserts) under increasing ethanol concentrations.

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.

A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: From biofuels and chemicals, to biocatalysis and bioremediation.

Metabolites, substrates and substrate impurities may be toxic to cells by damaging biological molecules, organelles, membranes or disrupting biological processes. Chemical stress is routinely encountered in bioprocessing to produce chemicals or fuels from renewable substrates, in whole-cell biocatalysis and bioremediation. Cells respond, adapt and may develop tolerance to chemicals by mechanisms only partially explored, especially for multiple simultaneous stresses.