Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

This study analyzed the cellular antioxidant and antiproliferative activities, as well as the phenolic composition of three Morchella conica Pers. cultures. Results showed that the free phenolic contents of the three Morchella conica Pers. ranged from 4.928 to 6.157 mg GAE/g DW and their bound phenolic contents ranged from 0.188 to 0.250 mg GAE/g DW. Polyphenols in M. conica Pers. were dominated by phenolic acids, particularly for gallic acid. The free phenolic extracts exhibited higher cellular antioxidant activity than the bound phenolic extracts. Free phenolics in M. conica Pers. cultured from Yunnan China showed the highest antiproliferative activity against HepG 2 cells, whereas bound phenolics in M. conica cultured from Tibet China showed the highest antiproliferative activity. Results confirmed that Morchella conica Pers. (growing in Yunnan China especially) could be a new source of natural antioxidant and a potential inhibitor for the growth of HepG 2 cells.