Selecting Better Biocatalysts by Complementing Recoded Bacteria.

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.

Prospective clinical evaluation of McCarey-Kaufman and organ culture cornea preservation media: 14-year follow-up.

PURPOSE: To compare the outcome of corneal grafts preserved in McCarey-Kaufman (MK) medium versus organ culture after penetrating keratoplasties. METHODS: Paired corneas were stored in McCarey medium for 2-44 hours (mean 21 hours) and in organ culture (OC) for 144 -240 hours (mean 192 hours). Penetrating keratoplasties were performed by 2 surgeons in 2 groups of patients with keratoconus, matched for age. Each pair was transplanted by the same surgeon using the same technique. Visual acuity, central corneal thickness, and central endothelial cell density were assessed at 166 +/- 7.8 months postoperatively. RESULTS: Nine pairs of patients were recovered for a long-term follow-up. The mean endothelial cell densities for the MK and OC groups were 611 +/- 155 and 683 +/- 168 cells/mm, respectively, which were not significantly different. A first rapid cell loss rate of 2.07% and 2.52% per month and a second slow of 0.78% and 0.69% per month were observed in the respective groups. Individual values of best corrected visual acuity were all the same (value 1.00) for both groups. Corneal thicknesses were, respectively, 571 +/- 52 and 540 +/- 35 microm and were significantly different (P = 0.013). CONCLUSIONS: After 14 years of penetrating keratoplasties performed with corneas stored in MK versus OC, no significant differences were observed in visual acuity, endothelial cell density, and cell loss. The observed thinner grafts after OC compared with MK could not be explained.