Polysaccharide-degrading Enzymes Research Articles

The mechanism by which Enteromorpha Linza polysaccharide promotes Bacillus subtilis growth and nitrate removal

In this study, we discussed the relationship between Entermorpha linza polysaccharide (EP) and Bacillus subtilis, which can transform nitrate. A sole carbon source experiment showed that Bacillus subtilis could utilize EP, and the bacterial density was maximally increased by 54.43% in the EP groups. The results of reducing sugar determination proved the secretion of polysaccharide-degrading enzymes. Scanning electron microscopy (SEM) showed that the EP groups had fewer spores and shrunken bacteria, indicating that EP could improve the growth environment and maintain bacterial integrity. Additionally, the ratios of periplasmic nitrate reductase (NAP), nitrite reductase (NIR), and dissimilatory nitrate reductase (D-NRase) in the EP groups were maximally increased by 107.22%, 84.70% and 36.10%, respectively. Transcriptome analysis further confirmed the above mentioned results. For example, the high expression of quorum sensing genes indicated that EP groups had higher bacterial density. Moreover, the high expression of antioxidant genes in the EP groups may be related to morphological integrity. Our study provides a basis for further discussion of the mechanism.

Non-starch polysaccharide-degrading enzymes may improve performance when included in wheat- but not maize-based diets fed to broiler chickens under subclinical necrotic enteritis challenge.

The present study investigated whether supplementing fibre-degrading enzymes can ameliorate the severity of subclinical necrotic enteritis (NE) in broiler chickens offered wheat- or maize-based diets. A total of 1,544 mixed-sex broiler chickens were assigned to 16 experimental treatments as a 2 × 2 × 4 factorial arrangement of treatments. The factors were the following: NE challenge, yes or no; diet type, wheat- or maize-based; and enzyme supplementation, control (no enzyme), family 10 xylanase (XYN10), family 11 xylanase (XYN11) or β-mannanase (MAN). Each treatment was replicated 6 times, with 16 birds per replicate pen. A three-way challenge × diet type × enzyme interaction occurred for body weight at 21 d of age (P = 0.025) and overall feed conversion ratio (P = 0.001). In the non-challenged birds fed the wheat-based diet, supplementing MAN increased d 21 body weight compared to the control. In challenged birds fed the maize-based diet, supplemental XYN11 impeded body weight and overall FCR compared to the control. Birds offered the maize-based diet presented heavier relative gizzard weights at both 16 and 21 d of age (P < 0.001) and reduced liveability (P = 0.046) compared to those fed the wheat-based diet. Enzyme supplementation reduced ileal and jejunal digesta viscosity at 16 d of age only in birds fed the wheat-based diet (P < 0.001). XYN11 increased ileal digesta viscosity in birds fed the maize-based diet, and MAN reduced it in birds fed the wheat-based diet at 21 d of age (P = 0.030). Supplementing XYN11 improved ileal soluble non-starch polysaccharides (NSP) digestibility in birds fed the wheat-based diet compared to non-supplemented birds (P < 0.001). Birds fed the wheat-based diet displayed a higher abundance of Bifidobacterium, Lactobacillus and Enterobacteriaceae and butyric acid in the caeca at 16 d of age compared to birds fed the maize-based diet (P < 0.05). In conclusion, supplemental XYN11 exacerbated the negative impact of NE on growth performance in birds fed the maize-based diet. Supplementing wheat-based diets with fibre-degrading enzymes ameliorates production losses induced by NE.

Non-starch polysaccharide degradation in the gastrointestinal tract of broiler chickens fed commercial-type diets supplemented with either a single dose of xylanase, a double dose of xylanase, or a cocktail of non-starch polysaccharide-degrading enzymes

The aim of this study was to examine non-starch polysaccharide (NSP) degradation in the gastrointestinal tract of chickens fed a range of commercial-type diets supplemented with a commercial dose of xylanase, a double dose of xylanase or a cocktail of NSP – degrading enzymes. Cobb 500 broilers (n = 1,080) were fed 12 dietary treatments; 4 diets with differing primary grain sources (barley, corn, sorghum, and wheat) and three different enzyme treatments (commercial recommended dose of xylanase (16,000 BXU/kg), a double dose of xylanase (32,000 BXU/kg) or an NSP-degrading enzyme cocktail (xylanase, β-glucanase, cellulase, pectinase, mannanase, galactanase, and arabinofuranosidase at recommended commercial levels). There were 108 pens, approximately 10 birds per pen, 9 replicates per dietary treatment. The diets were fed as 3 phases, starter (d 0–12), grower (d 12–23), and finisher (d 23–35). On bird age d 12, 23, and 35, performance (total pen body weight, feed intake, and feed conversion ratio corrected for mortality [cFCR]), litter and excreta dry matter content, and ileal and total tract soluble and insoluble NSP degradability and free oligosaccharide digestibility was determined. On d 35, the quantity of NSP in the gizzard, jejunum, ileum and excreta was determined. Results from this study showed that the double xylanase dose and NSP-ase cocktail had positive impacts on starter phase performance in birds fed the corn- and wheat-based diets. In the grower phase in birds fed the barley-based diet, these enzyme treatments improved cFCR and increased litter dry matter content. The NSP-ase cocktail had a negative impact on finisher phase cFCR in birds fed the sorghum-based diet. The double xylanase dose induced a positive impact on NSP degradability and free oligosaccharide digestibility. In conclusion, there appears to be advantages to feeding broilers a double xylanase dose, but lack of consistency when using an NSP-ase cocktail containing many enzymes.

First report of seaweed-associated yeast from Indonesia: Species composition and screening of their polysaccharides-degrading enzymes

Abstract. Sibero MT, Frederick EH, Sabdono A, Wijayanti DP, Pringgenies D, Radjasa OK, Zilda DS, Murwani R. 2022. First report of seaweed-associated yeast from Indonesia: Species composition and screening of their polysaccharides-degrading enzymes. Biodiversitas 23: 1408-1419. Yeast has been widely utilized in various industries due to its enzyme properties. Therefore, plenty of studies focus on exploring yeast biodiversity from diverse sources. However, there are limited reports regarding marine yeast biodiversity and its potential from Indonesia. This study aimed to isolate and identify the seaweed-associated yeast from Jepara in Central Java and Sepanjang Beach in Gunung Kidul, Yogyakarta, then examined their potential to produce extracellular polysaccharides-degrading enzymes (EPEs). Marine yeasts were isolated using standard marine agar (STD) and potato dextrose agar (PDA). All isolates were characterized by their salinity tolerance, morphology, and species confirmation using DNA barcoding. Three EPEs consisting of agarase, alginate-lyase, and carrageenase were screened using polysaccharides-enriched agar media. A specific agarase encoding gene was detected with specific primers. In total, 21 seaweed-associated yeast were successfully isolated from 6 seaweeds and noted as facultative marine yeast. The DNA barcoding study discovered that these yeasts belonged to 5 genera, consisted of Aureobasidium, Candida, Debaryomyces, Hortaea, and Rhodotorula. Moreover, Candida was noted as the most abundant genus (71.42%). It was noted that Hortaea werneckii MTJ.11 and MTJ.13 were positive for all enzymes; Aureobasidium melanogenum MTGK.31 gave a positive result for agarase and carrageenase; while [Candida] zeylanoides MTGK.23 only exhibited alginate-lyase activity. Unfortunately, none of the primers used to detect the presence of genes encoding polysaccharide-degrading enzymes were successfully amplified.

Effect of Glucose on Endo-xylanase and β-xylosidase Production by Fungi Isolated in Indonesia

Xylanases are widely produced by fungi, and the production of polysaccharide-degrading enzymes, in general, are usually subjected to carbon catabolite repression. In this work, the ability of several Indonesian indigenous fungi to produce endo-xylanase and β-xylosidase and their responses to glucose as a repressor were determined. Ten fungi were grown in a liquid medium supplemented with glucose as the repressor (0, 1%, 3%, and 5%), and the endo-xylanase and β-xylosidase productions were assayed. Aspergillus aculeatus FIG1 and A. oryzae KKB4 produced 3.85 and 0.70 U/mL of endo-xylanase, respectively, compared with other strains (0.22 U/mL or less). Trichoderma asperellum PK1J2, T. virens MLT2J2, A. aculeatus FIG1, T. asperellum MLT5J1, A. oryzae KKB4, and T. asperellum MLT3J2 produced 0.021–0.065 U/mL of β-xylosidase, whereas the other strains produced 0.013 U/mL or less of β-xylosidase. Adding 1% glucose to the growth medium can partially repress endo-xylanase production in A. aculeatus FIG1, T. asperellum PK1J2, and T. virens MLT4J1 and completely repress other strains. By adding 1% glucose, strains FIG1, PK1J2, and MLT4J1 suffered almost complete repression of β-xylosidase production, although such strains exhibited partial repression of endo-xylanase production. β-Xylosidase produced by the other strains showed complete repression by adding 1% glucose, except for A. aculeatus FIG1, A. tamarii FNCC 6151, and T. asperellum MLT1J1, which showed partial repression. Therefore, adding 3% glucose to the growth medium can result in complete repression of endo-xylanase and β-xylosidase productions in all strains examined.

Bioprospecting of cow's ruminal microbiota from a slaughterhouse in Ambarawa, Central Java, Indonesia

Abstract. Murwani R, Sibero MT, Silitonga PRA, Ambariyanto A. 2021. Bioprospecting of cow's ruminal microbiota from a slaughterhouse in Ambarawa, Central Java, Indonesia. Biodiversitas 22: 5030-5038. Ruminal microorganisms play essential roles in maintaining ruminant health. However, most studies focused only on ruminal lactic acid bacteria (LAB), although other ruminal microorganisms might have biological properties for biotechnological purposes. Therefore, the current study aimed to isolate ruminal bacteria (LAB and non-LAB) and fungi from ruminal material and conducted a bioprospecting study to understand their ability to produce antibacterial compounds and polysaccharide-degrading enzymes. The ruminal bacteria were isolated on MRS and ISP4 agar, while PDA was used to isolate the different fungi. The antibacterial property was tested against multidrug-resistant Escherichia coli and Salmonella enterica ser. Typhi. The ability to produce agarase, alginate-lyase, and carrageenase was screened. Prospective isolates were identified using DNA barcoding approach. Twelve bacteria were isolated using MRS agar, six from ISP4 agar, and four fungi from PDA. Among twelve bacteria from MRS agar, eleven were considered LAB, which consisted of Lactobacillus plantarum and Pediococcus acidilactici. Several classes of bacteria such as actinobacteria, firmicutes, ?-proteobacteria, and ?-proteobacteria were isolated during this study. In addition, three fungal classes, namely Saccharomycetes, Eurotiomycetes, and Mucoromycetes were also isolated. All bacteria from MRS agar were suggested to have potential compounds with antimicrobial activity, while all ruminal fungi exhibited potential sources of polysaccharide-degrading enzymes.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

Полисахаридразрушающие ферменты как агенты, диспергирующие бактериальные биопленки

Развитие бактериальных биопленок зависит от секреции и сохранения внеклеточных полисахаридов, или экзополисахаридов, которые являются основными компонентами внеклеточного полисахаридного вещества биопленок. Экзополисахариды внеклеточного полисахаридного вещества обеспечивают структурную стабильность биопленки, адгезию и агрегацию микроорганизмов, физическую и химическую защиту бактерий от действия противомикробных препаратов и эффекторов иммунной системы макроорганизма. Бактериальные клетки, расположенные в биопленке, защищены от антибактериальных эндо- и экзофакторов внеклеточным полимерным матриксом. Для инициирования диспергирования биопленок микроорганизмы наряду с другими ферментами используют специфические гликозидгидролазы, которые разрушают полисахариды бактериальных биопленок. Гликозидгидролазы реализуют свое действие через гидролиз гликозидных связей: амилазы расщепляют α-1,4-; целлюлазы — β-1,4-; β-галактозидазы — β-1,3-гликозидные связи. Основными гликозидгидролазами, которые обладают антибиопленочным действием, являются: α-лизоцим, амилазы, дисперсин B, целлюлазы, гиалуронидаза, α- и β-маннозидазы, альгинат-лиазы. Данные ферменты вызывают разрушение полисахаридных полимеров, способствуя высвобождению бактерий. Бактерии, которые лишились защиты полисахаридного каркаса, подвергаются воздействию антибактериальных агентов. С учетом того, что деградация экзополисахаридов биопленок гликозидгидролазами приводит к выраженному диспергированию бактерий, данный антибиопленочный метод лечения может представлять собой универсальный подход к терапии инфекций, протекающих с формированием биопленок. Медикаментозные методы диспергирования биопленок при помощи полисахаридразрушающих ферментов, без сомнения, расширят арсенал антибиопленочной терапии хронических и рецидивирующих бактериальных инфекций, особенно вызванных антибиотикорезистентными бактериями.

Ulvan polysaccharide-degrading enzymes: An updated and comprehensive review of sources category, property, structure, and applications of ulvan lyases

Ulvan, extracted from the marine green algae belonging to the genus Ulva, is mainly composed of four monosaccharides consisting of rhamnose, xylose, glucuronic acid and iduronic acid. It has great potential in the fields of food, pharmaceuticals and chemistry. It can be degraded by ulvan lyase with a β-elimination mechanism, which cleaves the β-glycosidic bond between sulfated rhamnose and glucuronic acid/iduronic acid, thereby producing an Ulva oligosaccharide with unsaturated bond. These oligosaccharides have several pleasing functions, including antioxidant, antiviral, antilipidic, antibacterial, and other effects. The latest CAZy data shows that ulvan lyases are composed of four families of lyases: PL24, PL25, PL28, and PL40. The structural features of some enzymes have been fully demonstrated, which is highly helpful in understanding their catalytic mechanisms. This review summarizes key sources, classifications, and key research advances in the biochemical properties of enzymes. In addition, its structural features and catalytic mechanism have been organized. Finally, we briefly summarized the potential of various active functional oligosaccharides produced by degrading ulvan in future applications. This detailed information can give us a better understanding of ulvan.

Metabolic Regulation of Sugar Assimilation for Lipid Production in Aspergillus oryzae BCC7051 through Comparative Transcriptome Perspective.

Simple SummaryOleaginous fungi are a promising candidate to produce microbial lipids as alternative sources for industrial applications. As lipids are intracellular metabolites with dynamic traits, the fungal ability in utilizing carbon sources for biomass and lipid production is significant in terms of production yield and economic feasibility. This study aimed to explore the metabolic regulation in lipogenesis of oleaginous Aspergillus oryzae BCC7051 at the transcriptional level. Through comparative transcriptome analysis, a set of differentially expressed genes (DEGs) between the xylose- and glucose-grown cultures (C5 and C6 cultures) at fast-growing and lipid-accumulating stages were identified and functionally categorized into transporter proteins and cellular processes. Combining with the growth and lipid phenotypes, the transcriptome results pointed to a crucial link between sugar assimilation, energy, lipid, and other metabolisms in A. oryzae for leveraging the metabolic flux from xylose to fatty acid and lipid biosynthesis in render the oleaginous features. This study provides a remarkable insight in guiding strain optimization and bioprocess development using renewable feedstocks from agroindustrial residues.Microbial lipid production with cost effectiveness is a prerequisite for the oleochemical sector. In this work, genome-wide transcriptional responses on the utilization of xylose and glucose in oleaginous Aspergillus oryzae were studied with relation to growth and lipid phenotypic traits. Comparative analysis of the active growth (t1) and lipid-accumulating (t2) stages showed that the C5 cultures efficiently consumed carbon sources for biomass and lipid production comparable to the C6 cultures. By pairwise comparison, 599 and 917 differentially expressed genes (DEGs) were identified in the t1 and t2 groups, respectively, in which the consensus DEGs were categorized into polysaccharide-degrading enzymes, membrane transports, and cellular processes. A discrimination in transcriptional responses of DEGs set was also found in various metabolic genes, mostly in carbohydrate, amino acid, lipid, cofactors, and vitamin metabolisms. Although central carbohydrate metabolism was shared among the C5 and C6 cultures, the metabolic functions in acetyl-CoA and NADPH generation, and biosynthesis of terpenoid backbone, fatty acid, sterol, and amino acids were allocated for leveraging biomass and lipid production through at least transcriptional control. This study revealed robust metabolic networks in the oleaginicity of A. oryzae governing glucose/xylose flux toward lipid biosynthesis that provides meaningful hints for further process developments of microbial lipid production using cellulosic sugar feedstocks.

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

ABSTRACTThe production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen.IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse “capsule depolymerases” which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.

Identification of genes involved in the formation of soluble dietary fiber in winter rye grain and their expression in cultivars with different viscosities of wholemeal water extract

The grain of rye (Secale cereale L.) used for baking contains a large amount of non-starch polysaccharides, making it an excellent component of functional foods. But rye grain intended for alcohol production and forage use should have a reduced content of these polysaccharides. A comprehensive parameter that can predict the best field of application for winter rye grain is the viscosity of its wholemeal water extract. However, our understanding of the genetic background underlying this key trait and associated features of rye grain is poor. By analyzing six Russian winter rye cultivars, we identified the most contrasting forms and characterized the peculiarities of their water-soluble carbohydrates capable of influencing the viscosity of water extracts. Then, using phylogenetic and transcriptomic analyses, we identified in the rye genome many genes encoding putative glycosyltransferases and glycosylhydrolases responsible for the synthesis and degradation of arabinoxylans, mixed-linkage glucans, cellulose, and some other polysaccharides. We determined the dynamics of mRNA abundance for these genes at three stages of kernel development. Comparisons of gene expression levels in two contrasting cultivars revealed specific members of multigene families that may serve as promising targets for manipulating non-starch polysaccharide content in rye grain. High-viscosity cultivars were characterized by up-regulation of many glycosyltransferases involved in the biosynthesis of arabinoxylans and other cell-wall polysaccharides, whereas low-viscosity cultivars showed up-regulation of several genes encoding polysaccharide-degrading enzymes.

Efficient chito-oligosaccharide utilization requires two TonB-dependent transporters and one hexosaminidase in Cellvibrio japonicus.

Chitin utilization by microbes plays a significant role in biosphere carbon and nitrogen cycling, and studying the microbial approaches used to degrade chitin will facilitate our understanding of bacterial strategies to degrade a broad range of recalcitrant polysaccharides. The early stages of chitin depolymerization by the bacteriumCellvibrio japonicushave been characterized and are dependent on one chitin-specific lytic polysaccharide monooxygenase and nonredundant glycoside hydrolases from the family GH18 to generate chito-oligosaccharides for entry into metabolism. Here, we describe the mechanisms for the latter stages of chitin utilization by C. japonicus with an emphasis on the fate of chito-oligosaccharides. Our systems biology approach combined transcriptomics and bacterial genetics using ecologically relevant substrates to determine the essential mechanisms for chito-oligosaccharide transport and catabolism in C. japonicus. Using RNAseq analysis we found a coordinated expression of genes that encode polysaccharide-degrading enzymes. Mutational analysis determined that the hex20B gene product, predicted to encode a hexosaminidase, was required for efficient utilization of chito-oligosaccharides. Furthermore, two gene loci (CJA_0353 and CJA_1157), which encode putative TonB-dependent transporters, were also essential for chito-oligosaccharides utilization. This study further develops our model of C. japonicuschitin metabolism and may be predictive for other environmentally or industrially important bacteria.

Novel Acinetobacter baumannii Myovirus TaPaz Encoding Two Tailspike Depolymerases: Characterization and Host-Recognition Strategy.

Acinetobacter baumannii, one of the most significant nosocomial pathogens, is capable of producing structurally diverse capsular polysaccharides (CPSs) which are the primary receptors for A. baumannii bacteriophages encoding polysaccharide-degrading enzymes. To date, bacterial viruses specifically infecting A. baumannii strains belonging to more than ten various capsular types (K types) were isolated and characterized. In the present study, we investigate the biological properties, genomic organization, and virus–bacterial host interaction strategy of novel myovirus TaPaz isolated on the bacterial lawn of A. baumannii strain with a K47 capsular polysaccharide structure. The phage linear double-stranded DNA genome of 93,703 bp contains 178 open reading frames. Genes encoding two different tailspike depolymerases (TSDs) were identified in the phage genome. Recombinant TSDs were purified and tested against the collection of A. baumannii strains belonging to 56 different K types. One of the TSDs was demonstrated to be a specific glycosidase that cleaves the K47 CPS by the hydrolytic mechanism.

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E.

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

Phylogenetic Distribution of Polysaccharide-Degrading Enzymes in Marine Bacteria.

Deconstruction is an essential step of conversion of polysaccharides, and polysaccharide-degrading enzymes play a key role in this process. Although there is recent progress in the identification of these enzymes, the diversity and phylogenetic distribution of these enzymes in marine microorganisms remain largely unknown, hindering our understanding of the ecological roles of marine microorganisms in the ocean carbon cycle. Here, we studied the phylogenetic distribution of nine types of polysaccharide-degrading enzymes in marine bacterial genomes. First, we manually compiled a reference sequence database containing 961 experimentally verified enzymes. With this reference database, we annotated 9,335 enzyme sequences from 2,182 high-quality marine bacterial genomes, revealing extended distribution for six enzymes at the phylum level and for all nine enzymes at lower taxonomic levels. Next, phylogenetic analyses revealed intra-clade diversity in the encoding potentials and phylogenetic conservation of a few enzymes at the genus level. Lastly, our analyses revealed correlations between enzymes, with alginate lyases demonstrating the most extensive correlations with others. Intriguingly, chitinases showed negative correlations with cellulases, alginate lyases, and agarases in a few genera. This result suggested that intra-genus lifestyle differentiation occurred many times in marine bacteria and that the utilization of polysaccharides may act as an important driver in the recent ecological differentiation of a few lineages. This study expanded our knowledge of the phylogenetic distribution of polysaccharide enzymes and provided insights into the ecological differentiation of marine bacteria.

Changes in cell wall components and polysaccharide-degrading enzymes in relation to differences in texture during sweetpotato storage root growth

Sweetpotato has special texture characteristics, which directly affect the eating quality and post-production processing quality of sweetpotato. To investigate the texture change mechanism of sweetpotato during the growth process, this study selected two varieties with significant differences in texture from 35 varieties. The storage roots were sampled at 50, 80, 110, and 140 days after planting. Measure the texture parameters, the cell wall composition content, cell wall-related enzyme activities and the expression of expansin genes of sweetpotato storage roots. The results show that the hardness, adhesiveness and chewiness parameters of ‘Yushu No 10′ were significantly lower than those of ‘Mianfen No 1′, they have significantly different texture properties. In terms of cell wall composition, the soluble pectin content of ‘Yushu No 10′ was more than twice that of ‘Mianfen No 1′, whereas the insoluble pectin content was lower than that of ‘Mianfen No 1′, with the cellulose content of ‘Yushu No 10′ being significantly higher than that of ‘Mianfen No 1′. In terms of cell wall-related enzymes, ‘Yushu No 10′ hardness gumminess and chewiness had a significant correlation with hemicellulose activity, and ‘Mianfen No 1′ had insignificant correlation with four cell wall-related enzymes. Expansin genes were also expressed differently during the various stages of root tubers expansin. The expressions of IbEXP1, IbEXP2 and IbEXPL1 were significantly correlated with the changes in cell wall component content, and were related to the qualitative structure changes. The research conclusion shows that the texture changes during the growth of sweetpotato are related to cell wall composition, cell wall-related enzyme activity changes, and the expression of expansin genes. This study provides theoretical guidance for in-depth study of texture changes of sweetpotato, post-harvest processing and utilization and quality improvement of storage roots.

Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium.

Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial factors economically challenging to its use. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge, enrichment strategies can be applied to develop microbial communities with an efficient CAZyme arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of the ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S rRNA sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of the ERAC disclosed 41 metagenome-assembled genomes (MAGs) harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The amino acid sequences of the majority of the predicted CAZymes (60% of the total) shared less than 90% identity with the sequences found in public databases. Additionally, a clostridial MAG identified in this study produced proteins during consortium development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism.IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries.

Microbial Community Dynamics During the Non-filamentous Fungi Growth-Based Fermentation Process of Miang, a Traditional Fermented Tea of North Thailand and Their Product Characterizations

Miang, a traditional fermented tea leaf (Camellia sinensis var. assamica) consumed in northern Thailand, was simulated in laboratory conditions using non-filamentous fungi process (NFP) and microbial community was periodically investigated for over 6 months of fermentation by both culture-dependent and -independent techniques. The viable cell numbers of lactic acid bacteria (LAB), yeast, and Bacillus enumerated by the culture-dependent technique markedly surged over 3 days of initial fermentation and then smoothly declined by the end of fermentation. LAB were found as the main microbial population throughout the fermentation period followed by yeast and Bacillus. High-throughput sequencing of microbial community during fermentation revealed that Firmicutes (86.9–96.0%) and Proteobacteria (4.0–12.4%) were the dominant bacterial phyla, whereas Ascomycota was found to be the main fungal phylum with an abundance of over 99% in the fungal community. The dominant bacterial family was Lactobacillaceae (39.7–79.5%) followed by Acetobacteraceae, Enterobacteriaceae, Bacillaceae, Aeromonadaceae, Staphylococcaceae, Moraxellaceae, Clostridiaceae, Exiguobacteraceae, Streptococcaceae, and Halomonadaceae. Meanwhile, the main fungal family was incertae sedis Saccharomycetales (75.6–90.5%) followed by Pichiaceae, Pleosporaceae, Botryosphaeriaceae, Davidiellaceae, Mycosphaerellaceae, and Saccharomycodaceae. In addition, Lactobacillus (29.2–77.2%) and Acetobacter (3.8–22.8%), and the unicellular fungi, Candida (72.5–89.0%) and Pichia (8.1–14.9%), were the predominant genera during the fermentation process. The profiles of physical and chemical properties such as Miang texture, pH, organic acids, polysaccharide-degrading enzyme activities, and bioactive compounds have rationally indicated the microbial fermentation involvement. β-Mannanase and pectinase were assumed to be the key microbial enzymes involved in the Miang fermentation process. Total tannin and total polyphenol contents were relatively proportional to the antioxidant activity. Lactic acid and butyric acid reached maximum of 50.9 and 48.9 mg/g dry weight (dw) at 9 and 63 days of fermentation, respectively. This study provided essential information for deeper understanding of the Miang fermentation process based on the chemical and biological changes during production.

One-step biohydrogen production from cassava pulp using novel enrichment of anaerobic thermophilic bacteria community

The aim of this study is to produce biohydrogen from untreated cassava pulp (CP) using a novel selected consortium of anaerobic thermophilic bacteria and the associated microbial communities were elucidated by metagenomic analysis. CP was chosen as a sole carbon source to enrich efficient hydrogen-producers from a total of 100 different composts and soil sediments under anaerobic dark fermentation at 60 °C. The stable enrichment culture was selected, base on CP degradation and hydrogen production. The selected consortium, namely BK16 from the residue of soil sediment around mangroves at Bangkhuntiean province, Thailand shows the highest hydrogen production yield (35.08 ml H 2 /g substrate) and hydrogen production rate (64.47 mL/day) in one-step. Besides, polysaccharide-degrading enzyme such as amylase, cellulase and xylanase of the BK16 were detected in culture supernatant at 70, 73 and 100 U/mg protein, respectively. In particular, the dominant microbes from selected consortium BK16 comprised of Clostridium thermopalmarium (61.2%) and Clostridium isatidis (25.3%) which have ability to degrade biomass and co-existed with acidogenesis bacteria, belong to the genus Thermoanaerobacterium (6.7%) and Fonticella tunisiensis (5.06%) to produced hydrogen as a consolidate bioprocess (CBP) system. The optimization condition for the hydrogen production of BK16 were at pH 7.0 and 10% (w/v) cassava pulp, in which 760 mL of H 2 /L was produced. Thus, these results revealed that the selected consortia BK16 can be used as an attractive candidate for direct hydrogen production, using cassava pulp as potential raw material for future bio-energy production. • The BK16 has isolated and identified the novel associated microbial communities. • Key biomass-degrading enzyme e.g., amylase, cellulase and xylanase were investigated. • One-step hydrogen production using non-pretreated cassava pulp were optimized.