Converting lignin components into a single product is a promising way to upgrade lignin. Here, an efficient biocatalyst was developed to selectively produce gallate from lignin components by integrating three main reactions: hydroxylation, O-demethylation, and aryl side-chain oxidation. A rationally designed hydroxylase system was first introduced into a gallate biodegradation pathway-blocked Rhodococcus opacus mutant so that gallate accumulated from protocatechuate and compounds in its upper pathways. Native and heterologous O-demethylation systems were then used, leading to multiple lignin-derived methoxy aromatics being converted to gallate. Furthermore, an aryl side-chain oxidase was engaged to broaden the substrate spectrum. Consequently, the developed biocatalyst showed that gallate yields as high as 0.407 and 0.630 g of gallate per gram of lignin when alkaline-pretreated lignin and base-depolymerized ammonia fiber explosion lignin were applied as substrates, respectively. These results suggested that this rationally developed biocatalyst enabled the lignin valorization process to be simple and efficient.

As multiple biochemical pathways exist in nature for lignin synthesis and degradation, lignin valorization through biological pathways has attracted much attention. cis,cis-Muconate, a common metabolic intermediate in lignin biodegradation, is an important platform chemical for the synthesis of a variety of polymers. In this work, Rhodococcus opacus PD630, which is efficient in the assimilation of aromatic compounds, was modified for the first time to accumulate cis,cis-muconate from lignin and lignin-derived aromatics. To achieve efficient genome modification for R. opacus PD630, a markerless gene deletion/insertion system was constructed based on a mutant phenylalanyl-tRNA synthase gene as a counter-selection marker. With the developed gene deletion/insertion system, several R. opacus PD630 mutants were successfully constructed for cis,cis-muconate production from lignin-derived aromatics using catechol and protocatechuate as two key funnel nodes. Results indicated the constructed strains could assimilate at least 15 types of lignin-derived aromatics and real lignin for cis,cis-muconate production. Moreover, both laccase addition and H 2 SO 4 + NaOH combinatorial pretreatment enhanced real lignin valorization to cis,cismuconate. Ultimately, 1.63 g/L cis,cis-muconate was produced using lignin from corn stover as the substrate by a fed-batch fermentation process. Overall, these results highlight the great potential of using R. opacus as a cell factory for valorizing lignin to cis,cis-muconate and other valuable products.

Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

Natural sweet protein monellin has a high sweetness and low calorie, suggesting its potential in food applications. However, due to its low heat and acid resistance, the application of monellin is limited. In this study, we show that the thermostability of monellin can be improved with no sweetness decrease by means of sequence, structure analysis, and site-directed mutagenesis. We analyzed residues located in the α-helix as well as an ionizable residue C41. Of the mutants investigated, the effects of E23A and C41A mutants were most remarkable. The former displayed significantly improved thermal stability, while its sweetness was not changed. The mutated protein was stable after 30 min incubation at 85°C. The latter showed increased sweetness and slight improvement of thermostability. Furthermore, we found that most mutants enhancing the thermostability of the protein were distributed at the two ends of α-helix. Molecular biophysics analysis revealed that the state of buried ionizable residues may account for the modulated properties of mutated proteins. Our results prove that the properties of sweet protein monellin can be modified by means of bioinformatics analysis, gene manipulation, and protein modification, highlighting the possibility of designing novel effective sweet proteins based on structure-function relationships.

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