Purification of biomevalonate from fermentation broth and conversion of biomevalonate into biomevalonolactone.

Mevalonate (MVA) is a key compound of living organisms including bacteria, plants, and humans. MVA and mevalonolactone (MVL), a lactonized form of MVA, are important for pharmaceutical, cosmeceutical, and biotechnological applications. Although (R, S)-MVA with 50% enantiomeric purity is mainly produced by chemical synthesis, recently, microbial fermentation processes for MVA production have been considered as an alternative to the chemical synthesis because of high enantiomeric purity [(R)-MVA] and high titer. In the present study, bio-MVA produced by a fermentative process was decolorized by a charcoal-based method and then chemically transformed into bio-MVL without byproducts by means of phosphoric acid as an acid catalyst. The final bio-MVL was (R)-MVL with over 99% enantiomeric purity according to 1H NMR analysis.

Incorporation of Unnatural Amino Acids in Response to the AGG Codon.

The biological protein synthesis system has been engineered to incorporate unnatural amino acid into proteins, and this has opened up new routes for engineering proteins with novel compositions. While such systems have been successfully applied in research, there remains a need to develop new approaches with respect to the wider application of unnatural amino acids. In this study, we reported a strategy for incorporating unnatural amino acids into proteins by reassigning one of the Arg sense codons, the AGG codon. Using this method, several unnatural amino acids were quantitatively incorporated into the AGG site. Furthermore, we applied the method to multiple AGG sites, and even to tandem AGG sequences. The method developed and described here could be used for engineering proteins with diverse unnatural amino acids, particularly when employed in combination with other methods.

The astaxanthin dideoxyglycoside biosynthesis pathway in Sphingomonas sp. PB304.

A major carotenoid in Sphingomonas sp. PB304, originally isolated from a river in Daejon City, South Korea, was identified as astaxanthin dideoxyglycoside. Gene clusters encoding the astaxanthin dideoxyglycoside biosynthetic enzymes were identified by screening Sphingomonas sp. PB304 fosmid libraries using degenerate probes that harbor highly conserved sequences from the Sphigomonas elodea-derived crtI and Nostoc sp. PCC 7120-dervied crtW genes. Selected positive gene clusters were fully sequenced and annotated, revealing genes encoding six putative carotenogenic enzymes: phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotene hydroxylase (CrtZ), carotene ketolase (CrtW), and glycosyltransferase (CrtX). All of the carotenogenic enzymes, except for CrtX, were functional in the recombinant host Escherichia coli expressing synthetic carotenogenic modules from Pantoea agglomerans. CrtX did not take up UDP-glucose or GDP-fucose as sugar substrates during the in vitro reaction. Although no direct experimental evidence was obtained for the function of Sphingomonas sp. PB304 CrtX, it can be categorized as a putative deoxyglycosyltransferase based on the presence of astaxanthin dideoxyglycoside in Sphingomonas sp. PB304, a putative corresponding gene in the carotenoid biosynthetic gene cluster, and high amino acid sequence homology to the existing glycosyltransferases. Therefore, we propose that astaxanthin dideoxyglycoside can be synthesized in Sphingomonas sp. PB304 via sequential reactions of six pathway enzymes, including CrtX on the phytoene intermediate.