Big data mining, rational modification, and ancestral sequence reconstruction inferred multiple xylose isomerases for biorefinery.

The isomerization of xylose to xylulose is considered the most promising approach to initiate xylose bioconversion. Here, phylogeny-guided big data mining, rational modification, and ancestral sequence reconstruction strategies were implemented to explore new active xylose isomerases (XIs) for Saccharomyces cerevisiae. Significantly, 13 new active XIs for S. cerevisiae were mined or artificially created. Moreover, the importance of the amino-terminal fragment for maintaining basic XI activity was demonstrated. With the mined XIs, four efficient xylose-utilizing S. cerevisiae were constructed and evolved, among which the strain S. cerevisiae CRD5HS contributed to ethanol titers as high as 85.95 and 94.76 g/liter from pretreated corn stover and corn cob, respectively, without detoxifying or washing pretreated biomass. Potential genetic targets obtained from adaptive laboratory evolution were further analyzed by sequencing the high-performance strains. The combined XI mining methods described here provide practical references for mining other scarce and valuable enzymes.

HDAC3 Knockdown Dysregulates Juvenile Hormone and Apoptosis-Related Genes in Helicoverpa armigera.

Insect development requires genes to be expressed in strict spatiotemporal order. The dynamic regulation of genes involved in insect development is partly orchestrated by the histone acetylation-deacetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although histone deacetylase 3 (HDAC3) is required for mice during early embryonic development, its functions in Helicoverpa armigera (H. armigera) and its potential to be used as a target of insecticides remain unclear. We treated H. armigera with HDAC3 siRNA and RGFP966, a specific inhibitor, examining how the HDAC3 loss-of-function affects growth and development. HDAC3 siRNA and RGFP966 treatment increased mortality at each growth stage and altered metamorphosis, hampering pupation and causing abnormal wing development, reduced egg production, and reduced hatching rate. We believe that the misregulation of key hormone-related genes leads to abnormal pupa development in HDAC3 knockout insects. RNA-seq analysis identified 2788 differentially expressed genes (≥two-fold change; p ≤ 0.05) between siHDAC3- and siNC-treated larvae. Krüppel homolog 1 (Kr-h1), was differentially expressed in HDAC3 knockdown larvae. Pathway-enrichment analysis revealed the significant enrichment of genes involved in the Hippo, MAPK, and Wnt signaling pathways following HDAC3 knockdown. Histone H3K9 acetylation was increased in H. armigera after siHDAC3 treatment. In conclusion, HDAC3 knockdown dysregulated juvenile hormone (JH)-related and apoptosis-related genes in H. armigera. The results showed that the HDAC3 gene is a potential target for fighting H. armigera.

Developing Rhodococcus opacus and Sphingobium sp. coculture systems for valorization of lignin-derived dimers.

Bioconversion is being regarded as a promising way for lignin valorization because it enables funneling diverse lignin components into single compounds, overcoming the heterogeneity of lignin. Although numerous lignin-derived aromatic monomers have been funneled to target compounds in previous studies, the bioconversion of low-molecular-weight lignin (LMW-lignin) fragments, for example, lignin-derived dimers, has been rarely systematically studied, impeding further conversion of lignin. In this study, coculture systems were designed and developed to funnel multiple lignin-derived dimers to cis, cis-muconate and gallate by combining lignin-derived dimers cleavage bacterium Sphingobium sp. and monomers conversion bacterium Rhodococcus opacus. With the developed coculture systems, ß-O-4 type dimer guaiacylglycerol-ß-guaiacyl ether, 4-O-5 type dimer 4,4'-dihydroxydiphenyl ether, ß-5 type dimer balanophonin, ß-ß type dimer pinoresinol, ß-1 type dimer 1,2-bis(4-hydroxy-3-methoxyphehyl)-1,3-propanediol and 5-5 type dimer 5,5'-dehydrodivanillate were converted to cis, cis-muconate. Additionally, the developed coculture systems also showed potential in conversion of lignin-derived dimers to gallate. The application of alkali lignin for cis, cis-muconate production further demonstrated the effectiveness of the designed coculture systems. Overall, the developed coculture systems are beneficial to lignin biological valorization, and also provide references for the valorization of other bio-resources.

Deciphering the metabolic distribution of vanillin in Rhodococcus opacus during lignin valorization.

Vanillin bioconversion is important for the biological lignin valorization. In this study, the obscure vanillin metabolic distribution in Rhodoccous opacus PD630 was deciphered by combining the strategies of intermediate detection, putative gene prediction, and target gene verification. The results suggest that approximately 10% (mol/mol) of consumed vanillin is converted to vanillic acid for further metabolism, and a large amount is converted to dead-end vanillyl alcohol in R. opacus PD630. Subsequently, five vanillin reductases were identified in R. opacus PD630, among which Pd630_LPD03722 product exhibited the greatest activity. With the detected metabolic distributions of vanillin, the conversion of vanillin to muconic acid was facilitated by deleting domestic vanillin reductase genes and introducing vanillin dehydrogenase from Sphingobium sp. SYK-6. Ultimately, the muconic acid yield from vanillin increased to 97.83% (mol/mol) from the initial 10% (mol/mol). Moreover, this study demonstrated the existence of vanillin reductases in Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum.

Valorization of lignin components into gallate by integrated biological hydroxylation, O-demethylation, and aryl side-chain oxidation.

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 pathway­blocked 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.

Microbial polyhydroxyalkanoate production from lignin by Pseudomonas putida NX-1.

Biological approaches play an important role in lignin valorization, whereas many issues in this area remain unclear. Herein, ligninolytic enzymes in Pseudomonas putida NX-1 were systematically unraveled based on genome sequence technology. Particularly, a dye-decolorizing peroxidase was systematically studied by heterologous expression, enzyme purification, and enzymatic characterization, which suggested it possessed activities on both synthetic dyes and lignin-derived aromatics. Moreover, a complete pathway for polyhydroxyalkanoate biosynthesis was annotated, and the polyhydroxyalkanoate biosynthesis capability of P. putida NX-1 was experimentally confirmed with lignin as the sole carbon source. Furthermore, the monomer compositions, molecular weights, and thermal properties of polyhydroxyalkanoate from glucose and lignin-derived aromatics were comprehensively determined by gas chromatography-mass spectrometry, gel permeation chromatography, differential scanning calorimetry, and thermogravimetric analysis. The results indicated that physical properties of polyhydroxyalkanoate prepared from glucose and lignin-derived aromatics were similar, which suggested lignin could be an alternative feedstock for polyhydroxyalkanoate production without compromising its quality.

Expression, purification and characterization of a novel double-sites mutant of the single-chain sweet-tasting protein monellin (MNEI) with both improved sweetness and stability.

The sweet protein monellin has high sweet potency with limited stability. In this study, 3 double-sites mutants (E2N/E23A, E2N/Y65R and E23A/Y65R) of the single-chain monellin (MNEI) were constructed. The proteins were expressed in E. coli BL21 and purified to homogeneity by nickel affinity chromatography with yields above 10 mg/L cell culture. Introduction of a sweeter mutant E2N into E23A or Y65R (E2N/E23A and E2N/Y65R) led to about 3-fold increase of sweetness, while addition of a more stable mutant E23A into E2N or Y65R (E2N/E23A and E23A/Y65R) resulted in improved thermal stability (about 10 °C). The results indicate that residues E2 and E23 mediate the sweetness and thermal stability of the protein, respectively. Multiple mutations of different residues (E2N/E23A) led to an additive performance with both improved sweetness and stability, suggesting that the sweetness and stability could be modulated by the independent molecular mechanism. The sweeter and thermal stable variant has a potential in further industrial applications.

Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

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.

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein, monellin, in Pichia pastoris with a constitutive GAPDH promoter and modified N-terminus.

OBJECTIVES: To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter. RESULTS: Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 µg/ml and 20 µg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains. CONCLUSIONS: Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering.

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.