Microbial tolerance engineering toward biochemical production: from lignocellulose to products.

Microbial metabolic engineering has been extensively studied for valuable chemicals synthesis, generating numerous laboratory-scale successes, and has demonstrated its potential to serve as a platform that enables large-scale manufacturing of many chemicals that are currently derived via chemical synthesis. However, the commercialization potential of microbial chemical production frequently suffers from low productivity and yields, where one key limiting factor is the inherently low tolerance of host cells against toxic compounds that are present and/or generated during biological processing. Consequently, various microbial engineering strategies have been devised to endow producer microbes with tolerance phenotypes that would be required for economically viable production of the desired chemicals. In this review, we discuss key microbial engineering strategies, devised primarily based on rational and evolutionary methodologies, that have been effective in improving cellular tolerance against fermentation inhibitors, metabolic intermediates, and valuable end-products derived from lignocellulose bioprocessing.

Cell-surface sensors for real-time probing of cellular environments.

The ability to explore cell signalling and cell-to-cell communication is essential for understanding cell biology and developing effective therapeutics. However, it is not yet possible to monitor the interaction of cells with their environments in real time. Here, we show that a fluorescent sensor attached to a cell membrane can detect signalling molecules in the cellular environment. The sensor is an aptamer (a short length of single-stranded DNA) that binds to platelet-derived growth factor (PDGF) and contains a pair of fluorescent dyes. When bound to PDGF, the aptamer changes conformation and the dyes come closer to each other, producing a signal. The sensor, which is covalently attached to the membranes of mesenchymal stem cells, can quantitatively detect with high spatial and temporal resolution PDGF that is added in cell culture medium or secreted by neighbouring cells. The engineered stem cells retain their ability to find their way to the bone marrow and can be monitored in vivo at the single-cell level using intravital microscopy.

Mimicking the inflammatory cell adhesion cascade by nucleic acid aptamer programmed cell-cell interactions.

Nature has evolved effective cell adhesion mechanisms to deliver inflammatory cells to inflamed tissue; however, many culture-expanded therapeutic cells are incapable of targeting diseased tissues following systemic infusion, which represents a great challenge in cell therapy. Our aim was to develop simple approaches to program cell-cell interactions that would otherwise not exist toward cell targeting and understanding the complex biology of cell-cell interactions. We employed a chemistry approach to engineer P- or L-selectin binding nucleic acid aptamers onto mesenchymal stem cells (MSCs) to enable them to engage inflamed endothelial cells and leukocytes, respectively. We show for the first time that engineered cells with a single artificial adhesion ligand can recapitulate 3 critical cell interactions in the inflammatory cell adhesion cascade under dynamic flow conditions. Aptamer-engineered MSCs adhered on respective selectin surfaces under static conditions >10 times more efficiently than controls including scrambled-DNA modified MSCs. Significantly, engineered MSCs can be directly captured from the flow stream by selectin surfaces or selectin-expressing cells under flow conditions (≤2dyn/cm²). The simple chemistry approach and the versatility of aptamers permit the concept of engineered cell-cell interactions to be generically applicable for targeting cells to diseased tissues and elucidating the biology of cell-cell interactions.