Distinct Adjacent Substrate Binding Pocket Regulates the Activity of a Decameric Feruloyl Esterase from Bacteroides thetaiotaomicron.

Understanding how the human gut microbiota contribute to the metabolism of dietary carbohydrates is of great interest, particularly those with ferulic acid (FA) decorations that have manifold health benefits. Here, we report the crystal structure of a decameric feruloyl esterase (BtFae) from Bacteroides thetaiotaomicron in complex with methyl ferulate (MFA), revealing that MFA is situated in a noncatalytic substrate binding pocket adjacent to the catalytic pocket. Molecular docking and mutagenesis studies further demonstrated that the adjacent pocket affects substrate binding in the active site and negatively regulates the BtFae activity on both synthetic and natural xylan substrates. Additionally, quantum mechanics (QM) calculations were employed to investigate the catalytic process of BtFae from substrate binding to product release, and identified TS_2 in the acylation step is rate-limiting. Collectively, this study unmasks a novel regulatory mechanism of FAE activity, which may contribute to further investigation of FA-conjugated polysaccharides metabolism in the human gut.

In vitro digestive properties and the bioactive effect of walnut green husk on human gut microbiota.

Introduction: Walnut green husk (WGH) is a waste byproduct from walnut industry. However, it is not well-known about its bioactive effect on human gut health. Methods: This study conducted in vitro digestion and fermentation experiments to study the bioactive effect of WGH. Results: Microbial fermentation was the primary mechanism to efficiently release phenolics and flavonoids, resulting in more excellent antioxidant capacities (DPPH, ABTS, and FRAP assays), which reached a highest value with 14.82 ± 0.01 mg VcE/g DW, 3.47 ± 0.01 mmol TE/g DW, and 0.96 ± 0.07 mmol FeSO4·7H2O/g DW, respectively. The surface microstructure of WGH became loose and fragmented after microbial fermentation. The analytical results of gut microbiota demonstrated that WGH could significantly increase the relative abundance of Proteobacteria in phylum level and Phascolarctobacterium in genus level while certain pro-inflammatory bacteria (such as Clostridium_sensu_stricto_1, Dorea, Alistipes, and Bilophila) was inhibited. Additionally, 1,373 differential metabolites were identified and enriched in 283 KEGG pathways. Of which some metabolites were significantly upregulated including ferulic acid, chlorogenic acid, umbelliferone, scopolin, muricholic acid, and so forth. Discussion: These results indicated that WGH could have antioxidant and anti-inflammatory activities in the human gut, which could improve the economical value of WGH in the food industry.

Construction of a Novel Vanillin-Induced Autoregulating Bidirectional Transport System in a Vanillin-Producing E. coli Cell Factory.

Vanillin is one of the world's most extensively used flavoring agents with high application value. However, the yield of vanillin biosynthesis remains limited due to the low efficiency of substrate uptake and the inhibitory effect on cell growth caused by vanillin. Here, we screened high-efficiency ferulic acid importer TodX and vanillin exporters PP_0178 and PP_0179 by overexpressing genes encoding candidate transporters in a vanillin-producing engineered Escherichia coli strain VA and further constructed an autoregulatory bidirectional transport system by coexpressing TodX and PP_0178/PP_0179 with a vanillin self-inducible promoter ADH7. Compared with strain VA, strain VA-TodX-PP_0179 can efficiently transport ferulic acid across the cell membrane and convert it to vanillin, which significantly increases the substrate utilization rate efficiency (14.86%) and vanillin titer (51.07%). This study demonstrated that the autoregulatory bidirectional transport system significantly enhances the substrate uptake efficiency while alleviating the vanillin toxicity issue, providing a promising viable route for vanillin biosynthesis.

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.

Structural analysis of receptor-like kinase SOBIR1 reveals mechanisms that regulate its phosphorylation-dependent activation.

Plant leucine-rich repeat (LRR) receptor-like kinases (RLKs) and LRR receptor-like proteins (RLPs) comprise a large family of cell surface receptors that play critical roles in signal perception and transduction. Both LRR-RLKs and LRR-RLPs rely on regulatory LRR-RLKs to initiate downstream signaling pathways. BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) and SUPPRESSOR OF BIR1-1 (SOBIR1) are important and extensively studied regulatory LRR-RLKs with distinct functions. Although the regulatory mechanism of BAK1 activation has been studied in detail, the activation mechanism of SOBIR1 remains poorly understood. Here, the crystal structures of the catalytically inactive kinase domain of SOBIR1 (SOBIR1-KD) from Arabidopsis thaliana were determined in complexes with AMP-PNP and Mg2+. The results show that SOBIR1-KD contains a uniquely long ß3-αC loop and adopts an Src-like inactive conformation with an unusual architecture at the activation segment, which comprises three helices. Biochemical studies revealed that SOBIR1 is transphosphorylated by BAK1 following its autophosphorylation via an intermolecular mechanism, and the phosphorylation of Thr529 in the activation segment and the ß3-αC loop are critical for SOBIR1 phosphorylation. Further functional analysis confirmed the importance of Thr529 and the ß3-αC loop for the SOBIR1-induced cell death response in Nicotiana benthamiana. Taken together, these findings provide a structural basis for the regulatory mechanism of SOBIR1 and reveal the important elements and phosphorylation events in the special stepwise activation of SOBIR1-KD, the first such processes found in regulatory LRR-RLKs.

Streptococcus mutans cell division protein FtsZ has higher GTPase and polymerization activities in acidic environment.

The acid tolerance of Streptococcus mutans plays an important role in its cariogenic process. Streptococcus mutans initiates a powerful transcriptional and physiological adaptation mechanism, eventually shielding the cellular machinery from acid damage and contributing to bacterial survival under acidic stress conditions. Although S. mutans contains complex regulatory systems, existing studies have shown that S. mutans, unlike Escherichia coli, cannot maintain a neutral intracellular environment. As the pH of the extracellular environment decreases, the intracellular pH decreases in parallel. There is insufficient knowledge regarding the acid resistance of the intracellular proteins of S. mutans, particularly when it comes to the key cytoskeletal division protein FtsZ. In this study, the data showed that S. mutans had similar cell division progress in acidic and neutral environments. The splitting position was in the middle of cells, and the cytoplasm was divided evenly in the acidic environment. Additionally, the tread milling velocity of S. mutans FtsZ in the middle of cells was not affected by the acidic environment. Streptococcus mutans FtsZ had higher GTPase activity in pH 6.0 buffer than in the neutral environment. Furthermore, the polymerization of S. mutans FtsZ in the acidic environment was more robust than that in the neutral environment. After two particular amino acids of S. mutans, FtsZ amino acids were mutated (E88K, L269K), the polymerization of S. mutans FtsZ in the acidic environment was significantly reduced. Overall, S. mutans FtsZ exhibited higher functional activity in pH 6.0 buffer in vitro. The acid resistance of S. mutans FtsZ is affected by its particular amino acids.

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.

Thermal and Acidic Treatments of Gluten Epitopes Affect Their Recognition by HLA-DQ2 in silico.

Celiac disease (CD) is a prevalent disorder with autoimmune features. Dietary exposure of wheat gluten (including gliadins and glutenins) to the small intestine activates the gluten-reactive CD4+ T cells and controls the disease development. While the human leukocyte antigen (HLA) is the single most important genetic factor of this polygenic disorder, HLA-DQ2 recognition of gluten is the major biological step among patients with CD. Gluten epitopes are often rich in Pro and share similar primary sequences. Here, we simulated the solution structures changes of a variety of gluten epitopes under different pH and temperatures, to mimic the fermentation and baking/cooking processes. Based on the crystal structure of HLA-DQ2, binding of differently processed gluten epitopes to DQ2 was studied in silico. This study revealed that heating and pH change during the fermentation process impact the solution structure of gluten epitope. However, binding of differently treated gluten epitope peptide (GEP) to HLA-DQ2 mainly depended on its primary amino acid sequence, especially acidic amino acid residues that play a pivotal role in their recognition by HLA-DQ2.

Rational Design for Broadened Substrate Specificity and Enhanced Activity of a Novel Acetyl Xylan Esterase from Bacteroides thetaiotaomicron.

Gut bacteria-derived enzymes play important roles in the metabolism of dietary fiber through enabling the hydrolysis of polysaccharides. In this study, we identified and characterized a 29 kDa novel acetyl xylan esterase, BTAxe1, from Bacteroides thetaiotaomicron VPI5482. Then, we solved the structure of BTAxe1 and performed the rational design. Mutants N65S and N65A increased the activities toward short-chain (pNPA, pNPB) to near four-fold, and gained the activities toward longer-chain substrate (pNPO). Molecular docking analysis showed that the mutant N65S had a larger substrate binding pocket than the wild type. Hydrolysis studies using natural substrates showed that either N65S or N65A showed higher activity of that of wild-type, yielding 131.31 and 136.09 mM of acetic acid from xylan. This is the first study on the rational design of gut bacteria-derived Axes with broadened substrate specificity and enhanced activity, which can be referenced by other acetyl esterases or gut-derived enzymes.

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.

[Research progresses in the biosynthesis of curcuminoids].

Curcuminoids are rare diketone compounds in plants and can be found in the rhizome of Curcuma longa as well as other Zingiberaceae and Araceae. Curcuminoids have been widely used in food and medical area owing to the yellow colors, as well as the antioxidant and many other pharmacological activities. Curcuminoids are a mixture of compounds containing curcumin, demethoxycurcumin and bisdemethoxycurcumin, which have distinct benzene ring substituents. Currently, curcuminoids are exclusively produced through plant extraction, which do not satisfy the meeting of the market demand. Empowered with new synthetic biology tools and metabolic engineering strategies, there is renewed interest in production of curcuminoids using microorganisms. Heterologous production of curcuminoids has been achieved using Escherichia coli, Yarrowia lipolytica, Pseudomonas putida and Aspergillus oryzae via engineering of curcuminoids biosynthesis pathway. In this review, we first describe the biological activities and various applications of curcuminoids. Next, we summarize the biosynthetic pathway of curcuminoids in Curcuma longa and discuss the catalytic mechanisms of curcumin synthases. Then, we thoroughly explore recent advances in the use of distinct microorganisms for the production of curcuminoids with a special focus on metabolic engineering strategies. Finally, we prospect the microbial production of curcuminoids by highlighting some promising techniques and approaches.

Bio-synthesis of food additives and colorants-a growing trend in future food.

Food additives and colorants are extensively used in the food industry to improve food quality and safety during processing, storage and packing. Sourcing of these molecules is predominately through three means: extraction from natural sources, chemical synthesis, and bio-production, with the first two being the most utilized. However, growing demands for sustainability, safety and "natural" products have renewed interest in using bio-based production methods. Likewise, the move to more cultured foods and meat alternatives requires the production of new additives and colorants. The production of bio-based food additives and colorants is an interdisciplinary research endeavor and represents a growing trend in future food. To highlight the potential of microbial hosts for food additive and colorant production, we focus on current advances for example molecules based on their utilization stage and bio-production yield as follows: (I) approved and industrially produced with high titers; (II) approved and produced with decent titers (in the g/L range), but requiring further engineering to reduce production costs; (III) approved and produced with very early stage titers (in the mg/L range); and (IV) new/potential candidates that have not been approved but can be sourced through microbes. Promising approaches, as well as current challenges and future directions will also be thoroughly discussed for the bioproduction of these food additives and colorants.

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.