Review of nomenclature and methods of analysis of polyethylene terephthalic acid hydrolyzing enzymes activity.

Enzymatic degradation of polyethylene terephthalic acid (PET) has been gaining increasing importance. This has resulted in a significant increase in the search for newer enzymes and the development of more efficient enzyme-based systems. Due to the lack of a standard screening process, screening new enzymes has relied on other assays to determine the presence of esterase activity. This, in turn, has led to various nomenclatures and methods used to describe them and measure their activity. Since all PET-hydrolyzing enzymes are α/ß hydrolases, they catalyze a serine nucleophilic attack and cleave an ester bond. They are lipases, esterases, cutinases and hydrolases. This has been used interchangeably, leading to difficulties while comparing results and evaluating progress. This review discusses the varied enzyme nomenclature being adapted, the different assays and analysis methods reported, and the strategies used to increase PET-hydrolyzing enzyme efficiency. A section on the various ways to quantify PET hydrolysis is also covered.

Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance.

Cyanobacteria hold promise as cell factories for the photoautotrophic conversion of carbon dioxide to useful chemicals. For the eventual commercial viability of such processes, cyanobacteria need to be engineered for (i) efficient channeling of carbon flux toward the product of interest and (ii) improved product tolerance, the latter being the focus of this study. We chose the recently reported, fast-growing, high light and CO2 tolerant cyanobacterium Synechococcus elongatus PCC 11801 for adaptive laboratory evolution. In two parallel experiments that lasted over 8400 h of culturing and 100 serial passages, S. elongatus PCC 11801 was evolved to tolerate 5 g/L n-butanol or 30 g/L 2,3-butanediol representing a 100% improvement in concentrations tolerated. The evolved strains retained alcohol tolerance even after being passaged several times without the alcohol stress suggesting that the changes were permanent. Whole genome sequencing of the n-butanol evolved strains revealed mutations in a number of stress responsive genes encoding translation initiation factors, RpoB and an ABC transporter. In 2,3-butanediol evolved strains, genes for ClpC, a different ABC transporter, glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase were found to be mutated. Furthermore, the evolved strains showed significant improvement in tolerance toward several other alcohols. Notably, the n-butanol evolved strain could tolerate up to 32 g/L ethanol, thereby making it a promising host for photosynthetic production of biofuels via metabolic engineering.