Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose.

Background: Sustainable alternatives for the production of fuels and chemicals are needed to reduce our dependency on fossil resources and to avoid the negative impact of their excessive use on the global climate. Lignocellulosic feedstock from agricultural residues, energy crops and municipal solid waste provides an abundant and carbon-neutral alternative, but it is recalcitrant towards microbial degradation and must therefore undergo extensive pretreatment to release the monomeric sugar units used by biofuel-producing microbes. These pretreatment steps can be reduced by using microbes such as Clostridium cellulolyticum that naturally digest lignocellulose, but this limits the range of biofuels that can be produced.

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.

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.

Inactivation of σF in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes σE and σG protein expression but does not block solvent formation.

Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation--both stationary-phase events--remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation.

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.

Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes.

Flow cytometry (FC) and FC-based cell sorting have been established as critical tools in modern cell and developmental biology. Yet, their applications in bacteria, especially in the multiparametric mode, remain limited. We argue that FC technologies have the potential to greatly accelerate the analysis and development of microbial complex phenotypes through applications of metabolic engineering, synthetic biology, and evolutionary engineering.

Development and application of flow-cytometric techniques for analyzing and sorting endospore-forming clostridia.

The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria.