Structural insights into the oligomeric effects on catalytic activity of a decameric feruloyl esterase and its application in ferulic acid production.

Oligomeric feruloyl esterase (FAE) has great application prospect in industry due to its potentially high stability and fine-tuned activity. However, the relationship between catalytic capability and oligomeric structure remains undetermined. Here we identified and characterized a novel, cold-adapted FAE (BtFae) derived from Bacteroides thetaiotaomicron. Structural studies unraveled that BtFae adopts a barrel-like decameric architecture unique in esterase families. By disrupting the interface, the monomeric variant exhibited significantly reduced catalytic activity and stability toward methyl ferulate, potentially due to its impact on the flexibility of the catalytic triad. Additionally, our results also showed that the monomerization of BtFae severely decreased the ferulic acid release from de-starched wheat bran and insoluble wheat arabinoxylan by 75 % and 80 %, respectively. Collectively, this study revealed novel connections between oligomerization and FAE catalytic function, which will benefit for further protein engineering of FAEs at the quaternary structure level for improved industrial applications.

In Vitro Fermentation of Beechwood Lignin-Carbohydrate Complexes Provides Evidence for Utilization by Gut Bacteria.

Lignin-carbohydrate complexes (LCCs) are emerging as a new and natural product with pharmacological and nutraceutical potential. It is uncertain, however, whether LCCs have a positive effect on the microbiota of the gut based on the current evidence. Here, the LCC extracted from beechwood (BW-LCC) was used as a substrate for in vitro fermentation. The lignin in BW-LCC consisted of guaiacyl (G) and syringyl (S) units, which are mainly linked by ß-O-4 bonds. After 24 h of in vitro fermentation, the pH had evidently declined. The concentrations of acetic acid and propionic acid, the two main short-chain fatty acids (SCFAs), were significantly higher than in the control group (CK). In addition, BW-LCC altered the microbial diversity and composition of gut microbes, including a reduction in the relative abundance of Firmicutes and an increase in the relative abundance of Proteobacteria and Bacteroidetes. The relative abundance of Escherichia coli-Shigella and Bacteroides were the most variable at the genus level. The genes of carbohydrate-active enzymes (CAZymes) also changed significantly with the fermentation and were related to the changes in microbes. Notably, the auxiliary actives (AAs), especially AA1, AA2, and AA3_2, play important roles in lignin degradation and were significantly enriched and concentrated in Proteobacteria. From this study, we are able to provide new perspectives on how gut microbes utilize LCC.

Coordinated regulation of Bacteroides thetaiotaomicron glutamate decarboxylase activity by multiple elements under different pH.

Glutamate decarboxylase catalyzes the conversion of glutamate to γ-aminobutyric acid, which plays a vital role in the gut-brain axis. Herein, a novel glutamate decarboxylase from Bacteroides thetaiotaomicron (BTGAD) was heterologously expressed. BTGAD possessed high catalytic efficiency at 60℃ and pH 3.6. As pH response, N-terminal sequence (NTS), C-terminal sequence (CTS), and ß-hairpin in BTGAD coordinately regulated its activity under different pH. NTS folded into a loop under acidic pH, and the truncation of NTS severely reduced its activity to 4.2%. While CTS occupied the active site under neutral pH and became disordered to release the inhibition effect under acidic conditions. The ß-hairpin, located near the active site, swung and formed open and closed conformations, which acted as an activity switch. This study provides the molecular basis for the coordinated regulation mechanism of BTGAD and lays a theoretical foundation for understanding the metabolism of dietary glutamate and its interaction relationships with the gut-brain axis.

Lactucin, a Bitter Sesquiterpene from Cichorium intybus, Inhibits Cancer Cell Proliferation by Downregulating the MAPK and Central Carbon Metabolism Pathway.

Lung cancer, especially adenocarcinoma, is the second most occurring and highest fatality-causing cancer worldwide. Many natural anticancer compounds, such as sesquiterpene lactones (SLs), show promising anticancer properties. Herein, we examined Lactucin, an SL from the plant Cichorium intybus, for its cytotoxicity, apoptotic-inducing, cell cycle inhibiting capacity, and associated protein expression. We also constructed a biotinylated Lactucin probe to isolate interacting proteins and identified them. We found that Lactucin stops the proliferation of A549 and H2347 lung adenocarcinoma cell lines while not affecting normal lung cell MRC5. It also significantly inhibits the cell cycle at G0/G1 stage and induces apoptosis. The western blot analysis shows that Lactucin downregulates the MAPK pathway, cyclin, and cyclin-dependent kinases, inhibiting DNA repair while upregulating p53, p21, Bax, PTEN, and downregulation of Bcl-2. An increased p53 in response to DNA damage upregulates p21, Bax, and PTEN. In an activity-based protein profiling (ABPP) analysis of A549 cell's protein lysate using a biotinylated Lactucin probe, we found that Lactucin binds PGM, PKM, and LDHA PDH, four critical enzymes in central carbon metabolism in cancer cells, limiting cancer cells in its growth; thus, Lactucin inhibits cancer cell proliferation by downregulating the MAPK and the Central Carbon Metabolism pathway.

Engineering the Active Site Pocket to Enhance the Catalytic Efficiency of a Novel Feruloyl Esterase Derived From Human Intestinal Bacteria Dorea formicigenerans.

The human gut microbiota play essential roles in metabolism and human health, especially by enzymatically utilizing dietary fiber that the host cannot directly digest and releasing functional components including short-chain fatty acids (SCFAs) and hydroxycinnamic acids (e.g., ferulic acid). In our previous study, seven potential feruloyl esterase (FAE) genes were identified from the gut microbiota. In the current work, one of the genes encoding a novel FAE (DfFAE) from Dorea formicigenerans of Firmicutes was bacterially expressed, purified and characterized. The 30.5 kDa type-A DfFAE has an optimum pH and temperature of 8.4 and 40 °C, respectively, exhibiting a higher substrate specificity toward short-chain acyl-ester substrate (pNPA). The AlphaFold2 based ab initio structural modeling revealed a five α-helices cap domain that shaped an unusually narrow and deep active site pocket containing a specific substrate access tunnel in DfFAE. Furthermore, rational design strategy was subjected to the active site pocket in an aim of improving its enzymatic activities. The mutants V252A, N156A, W255A, P149A, and P186A showed 1.8 to 5.7-fold increase in catalytic efficiency toward pNPA, while W255A also exhibited altered substrate preference toward long-chain substrate pNPO (45.5-fold). This study highlighted an unusual active site architecture in DfFAE that influenced its substrate selectivity and illustrated the applicability of rational design for enhanced enzymatic properties.

A reverse catalytic triad Asp containing loop shaping a wide substrate binding pocket of a feruloyl esterase from Lactobacillus plantarum.

Feruloyl esterase is an indispensable biocatalyst in food processing, pesticide and pharmaceutical industries, catalyzing the cleavage of the ester bond cross-linked between the polysaccharide side chain of hemicellulose and ferulic acid in plant cell walls. LP_0796 from Lactobacillus plantarum was identified as a feruloyl esterase that may have potential applications in the food industry, but the lack of the substrate recognition and catalytic mechanisms limits its application. Here, LP_0796 showed the highest activity towards methyl caffeate at pH 6.6 and 40 °C. The crystal structure of LP_0796 was determined at 2.5 Å resolution and featured a catalytic triad Asp195-containing loop facing the opposite direction, thus forming a wider substrate binding pocket. Molecular docking simulation and site-directed mutagenesis studies further demonstrated that in addition to the catalytic triad (Ser94, Asp195, His225), Arg125 and Val128 played essential roles in the function of the active site. Our data also showed that Asp mutation of Ala23 and Ile198 increased the catalytic efficiency to 4- and 5-fold, respectively. Collectively, this work provided a better understanding of the substrate recognition and catalytic mechanisms of LP_0796 and may facilitate the future protein design of this important feruloyl esterase.

The α-Helical Cap Domain of a Novel Esterase from Gut Alistipes shahii Shaping the Substrate-Binding Pocket.

The human gut microbiota regulates nutritional metabolism, especially by encoding specific ferulic acid esterases (FAEs) to release functional ferulic acid (FA) from dietary fiber. In our previous study, we observed seven upregulated FAE genes during in vitro fecal slurry fermentation using wheat bran. Here, a 29 kDa FAE (AsFAE) from Alistipes shahii of Bacteroides was characterized and identified as the type-A FAE. The X-ray structure of AsFAE has been determined, revealing a unique α-helical domain comprising five α-helices, which was first characterized in FAEs from the gut microbiota. Further molecular docking analysis and biochemical studies revealed that Tyr100, Thr122, Tyr219, and Ile220 are essential for substrate binding and catalytic efficiency. Additionally, Glu129 and Lys130 in the cap domain shaped the substrate-binding pocket and affected the substrate preference. This is the first report on A. shahii FAE, providing a theoretical basis for the dietary metabolism in the human gut.

Directed evolution of feruloyl esterase from Lactobacillus acidophilus and its application for ferulic acid production.

Producing ferulic acid (FA) from the natural substrate with feruloyl esterase is promising in industries, screening and engineering new enzymes with high efficiency to increase the FA yield is of great concern. Here, the feruloyl esterase of Lactobacillus acidophilus (FAELac) was heterologous expressed and the FAELac with different oligomerization states was separated. Interestingly, the activity of dimer was 37-fold higher than high-polymer. To further enhance the efficiency of FAELac, eight mutants were generated based on the simulated structure, of which Q198A, Q134T enhanced the catalytic efficiency by 5.4- and 4.3-fold in comparison with the wild type. Moreover, higher yields of FA (2.21, 6.60, and 1.67 mg/g substrate, respectively) were released by the mutants from de-starched wheat bran, insoluble wheat arabinoxylan, and steam-exploded corn stover. These results indicated that improving the purification process, engineering new FAELac and substrates bias studies hold great potential for increasing FA production yield.

The in vitro Effect of Fibers With Different Degrees of Polymerization on Human Gut Bacteria.

Human gut bacteria contribute significantly to human health and several studies have evaluated the effects of dietary fibers on human gut bacterial ecology. However, the relationship between different degrees of fiber polymerization and human gut bacteria is unknown. Here, we analyzed three fiber substrates with different degrees of polymerization, namely carboxymethylcellulose, ß-glucans, and galactooligosaccharides. To probe the in vitro influence of the degree of polymerization of the fiber on human gut bacteria, we measured the pH, air pressure, and short-chain fatty acid content of fecal fermentation supplemented with these fiber substrates, and sequenced the 16S ribosomal RNA genes of the microbial community in the fiber-treated fermentations. The butyric acid concentration was shown to decline with decreasing degree of polymerization of the fiber. Illumina Miseq sequencing indicated that the degree of polymerization might have an influence on human gut microbial diversity and abundance. Principal coordinate analysis unveiled a relationship between the degree of fiber polymerization and the gut bacterial community. Specific microbiota operational taxonomic units (OTUs) within the genera Escherichia-Shigella, Fusobacterium, and Dorea were proportional to the degree of fiber significantly, whereas OTUs within the genera Bifidobacterium, Streptococcus, and Lactobacillus were inversely correlated with the degree of polymerization. Correlation analysis between the fiber degree of polymerization and gut bacteria may demonstrate the effect of fibers on gut microbiota, and subsequently, on human health.

Site-directed mutagenesis of coenzyme-independent carotenoid oxygenase CSO2 to enhance the enzymatic synthesis of vanillin.

Vanillin is a popular flavoring compound and an important food additive. Owing to the consumer preference for inexpensive natural aroma flavors, vanillin production through a biotechnological pathway has become of great interest and commercial value in recent years. In this study, an enzymatic synthetic system for vanillin using a coenzyme-independent decarboxylase (FDC) and oxygenase (CSO2) cascade was reconstituted and optimized. This system produces a slightly higher production yield (40.20%) than the largest yield reported for immobilized FDC and CSO2 (35.00%) with ferulic acid as a substrate. It was previously reported that the low catalytic activity and thermal instability of CSO2 restrict the overall productivity of vanillin. In present study, site-directed mutagenesis was applied to rate-limiting oxygenase CSO2 to generate positive mutants. The production yields of mutants A49P (58.44%) and Q390A (65.29%) were 1.45- and 1.62-fold that of CSO2 wild type, respectively. The potential mechanism for enhanced vanillin production using A49P involved increased thermostability and catalytic efficiency, while that using Q390A was probably associated with a better thermostable performance and increased catalytic efficiency resulting from a larger entrance channel.

The Effect of Xylooligosaccharide, Xylan, and Whole Wheat Bran on the Human Gut Bacteria.

Wheat bran is a cereal rich in dietary fibers that have high levels of ferulic acid, which has prebiotic effects on the intestinal microbiota and the host. Herein we explored the effect of xylooligosaccharide, xylan, and whole wheat bran on the human gut bacteria and screened for potential ferulic acid esterase genes. Using in vitro fermentation, we analyzed the air pressure, pH-value, and short-chain fatty acid levels. We also performed 16S rRNA gene and metagenomic sequencing. A Venn diagram analysis revealed that 80% of the core operational taxonomic units (OTUs) were shared among the samples, and most of the xylooligosaccharide treatment core OTUs (319/333 OTUs) were shared with the other two treatments' core OTUs. A significant difference analysis revealed that the relative abundance of Dorea, Bilophila, and Sulfurovum in wheat bran treatment was higher than that in xylan and xylooligosaccharide treatments. The clusters of orthologous groups of proteins functional composition of all samples was similar to the microbiota composition of the control. Using metagenomic sequencing, we revealed seven genes containing the conserved residues, Gly-X-Ser-X-Gly, and the catalytic triad, Ser-His-Asp, which are thus potential ferulic acid esterase genes. All the results indicate that xylan and/or xylooligosaccharide, the main dietary fibers in wheat bran, plays a major role in in vitro fermentation by the human gut microbiota.

Citrus sinensis MYB transcription factors CsMYB330 and CsMYB308 regulate fruit juice sac lignification through fine-tuning expression of the Cs4CL1 gene.

Lignin is one of the most important components of the plant cell wall, and the expression and transcriptional regulation of lignin biosynthesis-related genes have been studied widely in Arabidopsis and other plants. Citrus fruit juice sacs often undergo lignification, particularly during fruit ripening and storage periods; however, the underlying genetic mechanisms have been little investigated. In this study, we isolated and identified CsMYB330 and CsMYB308 transcription factors, and found that their expression levels are significantly altered during the lignification of citrus fruit juice sacs. We found that CsMYB330 and CsMYB308 can recognize and bind AC elements in the Cs4CL1 promoter and finely regulate expression of the Cs4CL1 gene. In this regulatory process, CsMYB330 was identified as a transcriptional activator, whereas CsMYB308 appears to be a transcriptional repressor. In addition, using a transient assay, we demonstrated that expression of the Cs4CL1 gene is significantly altered in fruit juice sacs overexpressing these two transcription factors. These results indicate that the transcription factors CsMYB330 and CsMYB308 play important roles in the lignification of citrus fruit juice sacs and provide novel insights into the transcriptional regulation associated with fruit juice sac lignification.

Fungal Immunomodulatory Protein from Nectria haematococca Suppresses Growth of Human Lung Adenocarcinoma by Inhibiting the PI3K/Akt Pathway.

Lung cancer is a common disease that is associated with poor prognosis. Fungal immunomodulatory protein from Nectria haematococca (FIP-nha) has potential as a lung cancer therapeutic; as such, illuminating its anti-tumor mechanism is expected to facilitate novel treatment options. Here, we showed that FIP-nha affects lung adenocarcinoma growth ex vivo and in vivo. Comparative quantitative proteomics showed that FIP-nha negatively regulates PI3K/Akt signaling and induces cell cycle arrest, autophagy, and apoptosis. We further demonstrated that FIP-nha suppresses Akt phosphorylation, leading to upregulation of p21 and p27 and downregulation of cyclin B1, cyclin D1, CDK2, and CDK4 expression, ultimately resulting in G1/S and G2/M cell cycle arrest. Meanwhile, FIP-nha-induced PI3K/Akt downregulation promotes A549 apoptosis by increasing the expression ratio of Bax/Bcl-2 and c-PARP and autophagy by decreasing the phosphorylation of mTOR. Thus, we comprehensively revealed the anti-tumor mechanism of FIP-nha, which inhibits tumor growth by modulating PI3K/Akt-regulated cell cycle arrest, autophagy, and apoptosis, and provided the basis for further application of fungal immunomodulatory proteins, especially FIP-nha.

A new perspective of using sequential extraction: To predict the deficiency of trace elements during anaerobic digestion.

Trace elements were commonly used as additives to facilitate anaerobic digestion. However, their addition is often blind because of the complexity of reaction conditions, which has impeded their widespread application. Therefore, this study was conducted to evaluate deficiencies in trace elements during anaerobic digestion by establishing relationships between changes in trace element bioavailability (the degree to which elements are available for interaction with biological systems) and digestion performance. To accomplish this, two batch experiments were conducted. In the first, sequential extraction was used to detect changes in trace element fractions and then to evaluate trace element bioavailability in the whole digestion cycle. In the second batch experiment, trace elements (Co, Fe, Cu, Zn, Mn, Mo and Se) were added to the reaction system at three concentrations (low, medium and high) and their effects were monitored. The results showed that sequential extraction was a suitable method for assessment of the bioavailability of trace elements (appropriate coefficient of variation and recovery rate). The results revealed that Se had the highest (44.2%-70.9%) bioavailability, while Fe had the lowest (1.7%-3.0%). A lack of trace elements was not directly related to their absolute bioavailability, but was instead associated with changes in their bioavailability throughout the digestion cycle. Trace elements were insufficient when their bioavailability was steady or increased over the digestion cycle. These results indicate that changes in trace element bioavailability during the digestion cycle can be used to predict their deficiency.

Improving the methane yield of maize straw: Focus on the effects of pretreatment with fungi and their secreted enzymes combined with sodium hydroxide.

In order to improve the methane yield, the alkaline and biological pretreatments on anaerobic digestion (AD) were investigated. Three treatments were tested: NaOH, biological (enzyme and fungi), and combined NaOH with biological. The maximum reducing sugar concentrations were obtained using Enzyme T (2.20 mg/mL) on the 6th day. The methane yield of NaOH + Enzyme A was 300.85 mL/g TS, 20.24% higher than the control. Methane yield obtained from Enzyme (T + A) and Enzyme T pretreatments were 277.03 and 273.75 mL/g TS, respectively, which were as effective as 1% NaOH (276.16 mL/g TS) in boosting methane production, and are environmentally friendly and inexpensive biological substitutes. Fungal pretreatment inhibited methane fermentation of maize straw, 15.68% was reduced by T + A compared with the control. The simultaneous reduction of DM, cellulose and hemicellulose achieved high methane yields. This study provides important guidance for the application of enzymes to AD from lignocellulosic agricultural waste.

Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-ß-Xylanase from Caldicellulosiruptor owensensis.

Xylanases (EC 3.2.1.8) are a kind of enzymes degrading xylan to xylooligosaccharides (XOS) and have been widely used in a variety of industrial applications. Among them, xylanases from thermophilic microorganisms have distinct advantages in industries that require high temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10 xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90 °C with a half-life of about 1 h at 80 °C and exhibited highest activity at pH 7.0. The activity of CoXynA activity was affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA was proposed to be due to the increased overall protein rigidity resulting from the reduced length and fluctuation of Loop 7.

Expression and Characterization of Windmill Palm Tree (Trachycarpus fortunei) Peroxidase by Pichia pastoris.

Currently, commercial plant peroxidases are all native and are isolated from plants such as horseradish and soybean. No recombinant plant peroxidase products have been available on the commercial market. The gene encoding peroxidase was cloned from windmill palm tree leaves. The codon-optimized gene was transformed into Pichia pastoris for expression. The recombinant windmill palm tree peroxidase (rWPTP) expressed by P. pastoris showed high stability under pH 2-10 and temperatures up to 70 °C to many metallic salts and organic solvents. The substrate specificity of WPTP was determined, and among the substrates tested, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was most suitable for WPTP. The Michaelis constants with the substrates H2O2 and ABTS were 4.6 × 10-4 and 1.6 × 10-4 M, respectively. The rWPTP expressed in P. pastoris may be a suitable enzyme for the biosynthesis of polymers because of its high stability and activity under acidic conditions.

Methane Potential and Microbial Community Dynamics in Anaerobic Digestion of Silage and Dry Cornstalks: a Substrate Exchange Study.

Silage and dry are the two typical cornstalk forms. Either form could be used as substrate in biogas plants and might be replaced by another when shortage occurred. This study focused on the feeding sequence of these two kinds of feedstocks, aiming to discuss their specific methane potential (SMP). A 15-day hydraulic retention time was chosen for semi-continuous experiments based on the batch test results. In semi-continuous experiments, before and after feedstocks were exchanged, the significantly decreased and comparable SMPs of silage and dry cornstalks indicated that a basis of unstable digestion would result in incomplete methane release from the subsequent digestion. A higher similarity of bacterial community structure and greater quantity of bacteria were shown in acidified silage cornstalk digestion through band similarity analysis. Methanosaetaceae and methanomicrobiales were the predominant methanogens, and aceticlastic methanogenesis was the main route for methane production. The different feeding sequences affected the hydrolysis course and further influenced the methanogenic proliferation. Our work suggests that silage cornstalk digestion should be conducted before dry cornstalk digestion.

Enhancing anaerobic digestion of cotton stalk by pretreatment with a microbial consortium (MC1).

Microbial pretreatment is beneficial in some anaerobic digestion systems, but the consortia used to date have not been able to effectively increase methane production from cotton stalk. In this study, a thermophilic microbial consortium (MC1) was used for pretreatment in order to enhance biogas and methane production yields. The results indicated that the concentrations of soluble chemical oxygen demand and volatile organic products increased significantly in the early stages of pretreatment. Ethanol, acetic acid, propionic acid, and butyric acid were the predominant volatile organic products in the MC1 hydrolysate. Biogas and methane production yields from cotton stalk were significantly increased following MC1 pretreatment. In addition, the methane production rate from the treated cotton stalk was greater than that from the untreated sample.

Comparison and evaluation of concurrent saccharification and anaerobic digestion of Napier grass after pretreatment by three microbial consortia.

Napier grass is potentially a viable feedstock for biofuel production. The present study investigated biological pretreatment of Napier grass by three microbial consortia followed by saccharification and anaerobic digestion. The pretreatment efficiencies of three microbial consortia were compared in terms of degradation ability, saccharide and biogas yield. The lignocellulose loss rates of Napier grass varied largely. The biomass pretreated by the consortium WSD-5 gave 43.4% and 66.2% total sugar yield under low and moderate loadings of commercial enzyme mixtures, while the highest yield was 83.2% pretreated by the consortium MC1 under a high enzyme loading. The maximum methane yield of pretreated samples by the consortia MC1, WSD-5 and XDC-2 were 259, 279, 247ml/g VS, respectively, which were 1.39, 1.49 and 1.32times greater than the values of the untreated controls. This study showed that pretreatments by MC1, WSD-5 and XDC-2 were capable of significantly enhancing both the saccharide and methane yields from Napier grass.