Transcriptional analysis and adaptive evolution of Escherichia coli strains growing on acetate.

Eighteen strains of Escherichia coli were compared for maximum specific growth rate (µ MAX) on 85 mM acetate as the sole carbon source. The C strain ATCC8739 had the greatest growth rate (0.41 h(-1)) while SCS-1 had the slowest growth rate (0.15 h(-1)). Transcriptional analysis of three of the strains (ATCC8739, BL21, SMS-3-5) was conducted to elucidate why ATCC8739 had the greatest maximum growth rate. Seventy-one genes were upregulated 2-fold or greater in ATCC8739, while 128 genes were downregulated 2-fold or greater in ATCC8739 compared to BL21 and SMS-3-5. To generate a strain that could grow more quickly on acetate, ATCC8739 was cultured in a chemostat using a progressively increasing dilution rate. When the dilution rate reached 0.50 h(-1), three isolated colonies each grew faster than ATCC8739 on 85 mM acetate, with MEC136 growing the fastest with a growth rate of 0.51 h(-1), about 25 % greater than ATCC8739. Transcriptional analysis of MEC136 showed that eight genes were downregulated 2-fold or greater and one gene was upregulated 2-fold or greater compared to ATCC8739. Genomic sequencing revealed that MEC136 contained a single mutation, causing a serine to proline change in amino acid 266 of RpoA, the α subunit of the RNA polymerase core enzyme. The 260-270 amino acid region of RpoA has been shown to be a key region of the protein that affects the interaction of the α subunit of the RNA polymerase core enzyme with several global transcriptional activators, such as CRP and FNR.

Microbial removal of acetate selectively from sugar mixtures.

Acetic acid is an unavoidable constituent of the biomass hydrolysates generated from acetylated hemicellulose and lignin, and acetate affects the performance of microbes used to convert these hydrolysates into biofuels or other biochemicals. In this study, acetate was selectively removed from synthetic mixtures of glucose and xylose using metabolically engineered Escherichia coli strains having mutations in the glucose phosphotransferase system (PTS) genes (ptsG, manZ, crr), glucokinase (glk), and xylose (xylA). In batch culture, ALS1060 (ptsG manZ glk xylA) consumed exclusively acetate to depletion, and then consumed the two sugars only at a very slow rate (a growth rate of about 0.01 h(-1)). We also examined the effects of an additional knockout of either malX, fruA, fruB, bglF, or crr, genes that are involved in other PTSs, and a batch process using KD840 (ptsG manZ glk crr xylA) demonstrated a further reduction in glucose or xylose consumption by E. coli. These results demonstrate the feasibility of using a substrate-selective approach for the pre-treatment of biomass hydrolysate for microbial processes.

Continuous-flow Ferrohydrodynamic Sorting of Particles and Cells in Microfluidic Devices.

A new sorting scheme based on ferrofluid hydrodynamics (ferrohydrodynamics) was used to separate mixtures of particles and live cells simultaneously. Two species of cells, including Escherichia coli and Saccharomyces cerevisiae, as well as fluorescent polystyrene microparticles were studied for their sorting throughput and efficiency. Ferrofluids are stable magnetic nanoparticles suspensions. Under external magnetic fields, magnetic buoyancy forces exerted on particles and cells lead to size-dependent deflections from their laminar flow paths and result in spatial separation. We report the design, modeling, fabrication and characterization of the sorting device. This scheme is simple, low-cost and label-free compared to other existing techniques.