Genomic Analysis to Elucidate the Lignocellulose Degrading Capability of a New Halophile Robertkochia solimangrovi.

Robertkochia solimangrovi is a proposed marine bacterium isolated from mangrove soil. So far, the study of this bacterium is limited to taxonomy only. In this report, we performed a genomic analysis of R. solimangrovi that revealed its lignocellulose degrading ability. Genome mining of R. solimangrovi revealed a total of 87 lignocellulose degrading enzymes. These enzymes include cellulases (GH3, GH5, GH9 and GH30), xylanases (GH5, GH10, GH43, GH51, GH67, and GH115), mannanases (GH2, GH26, GH27 and GH113) and xyloglucanases (GH2, GH5, GH16, GH29, GH31 and GH95). Most of the lignocellulolytic enzymes encoded in R. solimangrovi were absent in the genome of Robertkochia marina, the closest member from the same genus. Furthermore, current work also demonstrated the ability of R. solimangrovi to produce lignocellulolytic enzymes to deconstruct oil palm empty fruit bunch (EFB), a lignocellulosic waste found abundantly in palm oil industry. The metabolic pathway taken by R. solimangrovi to transport and process the reducing sugars after the action of lignocellulolytic enzymes on EFB was also inferred based on genomic data. Collectively, genomic analysis coupled with experimental studies elucidated R. solimangrovi to serve as a promising candidate in seawater based-biorefinery industry.

Genome-wide identification and characterization of active ingredients related ß-Glucosidases in Dendrobium catenatum.

BACKGROUND: Dendrobium catenatum/D. officinale (here after D. catenatum), a well-known economically important traditional medicinal herb, produces a variety of bioactive metabolites including polysaccharides, alkaloids, and flavonoids with excellent pharmacological and clinical values. Although many genes associated with the biosynthesis of medicinal components have been cloned and characterized, the biosynthetic pathway, especially the downstream and regulatory pathway of major medicinal components in the herb, is far from clear. ß-glucosidases (BGLUs) comprise a diverse group of enzymes that widely exist in plants and play essential functions in cell wall modification, defense response, phytohormone signaling, secondary metabolism, herbivore resistance, and scent release by hydrolyzing ß-D-glycosidic bond from a carbohydrate moiety. The recent release of the chromosome-level reference genome of D. catenatum enables the characterization of gene families. Although the genome-wide analysis of the BGLU gene family has been successfully conducted in various plants, no systematic analysis is available for the D. catenatum. We previously isolated DcBGLU2 in the BGLU family as a key regulator for polysaccharide biosynthesis in D. catenatum. Yet, the exact number of DcBGLUs in the D. catenatum genome and their possible roles in bioactive compound production deserve more attention. RESULTS: To investigate the role of BGLUs in active metabolites production, 22 BGLUs (DcBGLU1-22) of the glycoside hydrolase family 1 (GH1) were identified from D. catenatum genome. Protein prediction showed that most of the DcBGLUs were acidic and phylogenetic analysis classified the family into four distinct clusters. The sequence alignments revealed several conserved motifs among the DcBGLU proteins and analyses of the putative signal peptides and N-glycosylation site revealed that the majority of DcBGLU members dually targeted to the vacuole and/or chloroplast. Organ-specific expression profiles and specific responses to MeJA and MF23 were also determined. Furthermore, four DcBGLUs were selected to test their involvement in metabolism regulation. Overexpression of DcBGLU2, 6, 8, and 13 significantly increased contents of flavonoid, reducing-polysaccharide, alkaloid and soluble-polysaccharide, respectively. CONCLUSION: The genome-wide systematic analysis identified candidate DcBGLU genes with possible roles in medicinal metabolites production and laid a theoretical foundation for further functional characterization and molecular breeding of D. catenatum.

Current Status of Mining, Modification, and Application of Cellulases in Bioactive Substance Extraction.

Cellulases have been used to extract bioactive ingredients from medical plants; however, the poor enzymatic properties of current cellulases significantly limit their application. Two strategies are expected to address this concern: (1) new cellulase gene mining strategies have been promoted, optimized, and integrated, thanks to the improvement of gene sequencing, genomic data, and algorithm optimization, and (2) known cellulases are being modified, thanks to the development of protein engineering, crystal structure data, and computing power. Here, we focus on mining strategies and provide a systemic overview of two approaches based on sequencing and function. Strategies based on protein structure modification, such as introducing disulfide bonds, proline, salt bridges, N-glycosylation modification, and truncation of loop structures, have already been summarized. This review discusses four aspects of cellulase-assisted extraction. Initially, cellulase alone was used to extract bioactive substances, and later, mixed enzyme systems were developed. Physical methods such as ultrasound, microwave, and high hydrostatic pressure have assisted in improving extraction efficiency. Cellulase changes the structure of biomolecules during the extraction process to convert them into effective ingredients with better activity and bioavailability. The combination of cellulase with other enzymes and physical technologies is a promising strategy for future extraction applications.

Functional characterizations of ß-glucosidases involved in aroma compound formation in tea (Camellia sinensis).

Tea (Camellia sinensis) aroma is an important factor affecting tea quality. Many tea aroma compounds are present as glycosidically conjugated forms in tea leaves, and can be hydrolyzed by ß-glucosidase (ß-Glu) and ß-primeverosidase to release free tea aromas. ß-Primeverosidase has been identified and functionally characterized, while ß-Glu has not been identified in tea leaves. In the present study, we established a yeast expression system to recombine CsGH1BG1, CsGH3BG1, and CsGH5BG1, which belonged to GH1, GH3, and GH5 families in plants, respectively. These three recombinant Csß-Glus hydrolyzed the ß-glucopyranosidically conjugated aromas to form free aromas, suggesting that there was no specific Csß-Glus for the hydrolysis of ß-glucopyranosidically conjugated aromas in vitro. Furthermore, subcellular localization of the Csß-Glus indicated that CsGH1BG1 and CsGH3BG1 were located in the cytosol and vacuole, respectively, while CsGH5BG1 was located in the cell wall. This suggested that CsGH1BG1 and CsGH3BG1 might be responsible for the hydrolysis of ß-glucopyranosidically conjugated aromas in tea leaves during the tea manufacturing process. This study provides the first evidence of Csß-Glus in tea leaves, and will advance understanding of tea aroma formation.

Chaotropic heat treatment resolves native-like aggregation of a heterologously produced hyperthermostable laminarinase.

Production of hyperthermostable enzymes in mesophilic hosts frequently causes undesired aggregation of these proteins. During production of Pyrococcus furiosus endo-ß-1,3 glucanase (LamA) in Escherichia coli, soluble and insoluble species form. Here, the authors address the composition of this mixture, including the nature of LamA conformers, and establish a method to increase the yield of native monomer. With gel electrophoresis, size-exclusion chromatography, light scattering, circular dichroism and enzyme kinetics the authors show that approximately 50 % of heterologously produced LamA is soluble, and that 40 % of this fraction constitutes native-like oligomers and non-native monomers. Soluble oligomers display, like native LamA monomer, substrate inhibition, although with poor activity. Treatment of soluble oligomers with 3 M guanidinium hydrochloride at 80 °C yields up to 75 % properly active monomer. Non-native monomer shows low specific activity without substrate inhibition. Incubating non-native monomer with 3 M guanidinium hydrochloride at 80 °C causes formation of 25 % native LamA. Also, a large amount of insoluble LamA aggregates can be converted into soluble native monomer by application of this procedure. Thus, chaotropic heat treatment can improve the yield and quality of hyperthermostable proteins that form aberrant species during production in E. coli.

Citrobacter freundii as a test platform for recombinant cellulose degradation systems.

Cellulosic biomass represents a huge reservoir of renewable carbon, but converting it into useful products is challenging. Attempts to transfer cellulose degradation capability to industrially useful micro-organisms have met with limited success, possibly due to poorly understood synergy between multiple cellulases. This is best studied by co-expression of many combinations of cellulases and associated proteins. Here, we describe the development of a test platform based on Citrobacter freundii, a cellobiose-assimilating organism closely related to Escherichia coli. Standard E. coli cloning vectors worked well in Cit. freundii. Expression of cellulases CenA and Cex of Cellulomonas fimi in Cit. freundii gave recombinant strains which were able to grow at the expense of cellulosic filter paper or microcrystalline cellulose (Avicel) in a mineral medium supplemented with a small amount of yeast extract. Periodic physical agitation of the cultures was highly beneficial for growth at the expense of filter paper. This provides a test platform for the expression of combinations of genes encoding biomass-degrading enzymes to develop effective genetic cassettes for degradation of different biomass streams. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofuels have been shown to be the best sustainable and alternative source of fuel to replace fossil fuels. Of the different types of feedstocks used for producing biofuels, lignocellulosic biomass is the most abundant. Converting this biomass to useful products has met with little success. Different approaches are being used and microbial platforms are the most promising and sustainable method. This study shows that Citrobacter freundii is a better test platform than Escherichia coli for testing various combinations of cellulases for the development of microbial systems for biomass conversion.

Microbacterium rhizomatis sp. nov., a ß-glucosidase-producing bacterium isolated from rhizome of Korean mountain ginseng.

A novel Gram-staining-positive, rod-shaped bacterium, designated DCY100(T), was isolated from rhizome of mountain ginseng root in Hwacheon mountain, Gangwon province, Republic of Korea. The 16S rRNA gene sequence analysis showed that strain DCY100(T) belonged to the genus Microbacterium and was most closely related to Microbacterium ginsengisoli KCTC 19189(T) (97.9%), Microbacterium lacus JCM 15575(T) (97.2%) and Microbacterium invictum DSM 19600(T) (97.1%). The major menaquinones were MK-11 and MK-12. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid. The major fatty acids (>10.0%) were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The cell-wall peptidoglycan contained the amino acids ornithine, alanine, glutamic acid and glycine; whole-cell sugars consisted of glucose, galactose, rhamnose and ribose. The DNA G+C content was 63.6 ± 0.7 mol%. The DNA-DNA hybridization relatedness values between strain DCY100(T) and Microbacterium ginsengisoli KCTC 19189(T), Microbacterium lacus JCM 15575(T) and Microbacterium invictum DSM 19600(T) were 36.2 ± 0.4, 22.0 ± 3.0 and 15.3 ± 1.8%, respectively. On the basis of phenotypic, chemotaxonomic and genotypic analyses, the isolate is classified as a representative of a novel species in the genus Microbacterium, for which the name Microbacterium rhizomatis DCY100(T) is proposed. The type strain is DCY100(T) ( = KCTC 39529(T) = JCM 30598(T)).

Streptomyces panaciradicis sp. nov., a ß-glucosidase-producing bacterium isolated from ginseng rhizoplane.

A Gram-staining-positive actinobacterium, designated strain 1MR-8(T), was isolated from the rhizoplane of ginseng and its taxonomic status was determined using a polyphasic approach. The isolate formed long chains of spores that were straight, cylindrical and smooth-surfaced. Strain 1MR-8(T) grew at 10-37 °C (optimum 28 °C), whilst no growth was observed at 45 °C. The pH range for growth was 4.0-11.0 (optimum pH 6.0-8.0) and the NaCl range for growth was 0-7% (w/v) with optimum growth at 1% (w/v). Strain 1MR-8(T) had cell-wall peptidoglycans based on ll-diaminopimelic acid. Glucose, mannose and ribose were the whole-cell sugars. The predominant isoprenoid quinones were MK-9 (H4), MK-9 (H6) and MK-9 (H8) and the major fatty acids were anteiso-C(15:0), iso-C(15:0), anteiso-C(17:0) and iso-C(16:0). 16S rRNA gene sequencing studies showed that the novel strain was closely related to the type strains of Streptomyces caeruleatus GIMN4(T), Streptomyces curacoi NRRL B-2901(T), Streptomyces capoamus JCM 4734(T) and Streptomyces coeruleorubidus NBRC 12761(T) with similarities of 98.8%. However, DNA-DNA relatedness, as well as physiological and biochemical analyses, showed that strain 1MR-8(T) could be differentiated from its closest phylogenetic relatives. It is proposed that this strain should be classified as a representative of a novel species of the genus Streptomyces, with the suggested name Streptomyces panaciradicis sp. nov. The type strain is 1MR-8(T) ( = KACC 17632(T) = NBRC 109811(T)).

A useful method integrating production and immobilization of recombinant cellulase.

The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase (celA), cellobiohydrolase (celK), and ß-glucosidase (gls) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli, each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose-hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

The porcine gut microbial metagenomic library for mining novel cellulases established from growing pigs fed cellulose-supplemented high-fat diets.

The porcine gut microbiome is a novel genomic resource for screening cellulose-degrading enzymes. A plasmid metagenomic expression library was constructed from the hindgut microbiota of 6 Yorkshire growing pigs (25 to 40 kg) fed a high-fat basal diet supplemented with 10% Solka-Floc for 28 d. Fresh cecal and colonic digesta samples were collected, flash-frozen in liquid N, and stored under -80°C. Metagenomic DNA was extracted, mechanically sheared, and cleaned to remove small DNA fragments (<1.0 kb). The resulting DNA fragments were subjected to blunt-end polishing, fractionation, and purification by using commercial kits. The end-modified DNA fragments were ligated to pCR4Blunt-TOPO vector and transformed into competent Escherichia coli TOPO10 cells. Metagenomic plasmid libraries were screened for carboxymethyl cellulolytic activities by using lysogeny broth agar plates. The average insert size of the resulting library was approximately 4.2 kb. Screening for the ability to hydrolyze carboxymethyl cellulose yielded 14 positive colonies, giving an estimated 430 Mb of metagenomic DNA in the approximately 102,000 E. coli clones with an overall hit rate of 0.14%. The 11 assembled insert sequences included 4 function-related gene clusters, and a total of 18 putative carbohydrate active enzyme genes were identified. This included genes encoding 11 cellulases, 4 hemicellulases, 1 polygalacturonas, 1 glycoside hydrolase family 26 mannanase-family 5 cellulase chimeric enzyme gene, and 1 cellobiose phosphorylase. In conclusion, the coupling of functional metagenomic mining with biochemical characterization of fiber-degrading enzymes is a powerful strategy for exploring the enzymological underpinnings of the anaerobic fermentation of dietary fiber in the complex animal gut environment.

Mutagenesis and genotypic characterization of Aspergillus niger FCBP-02 for improvement in cellulolytic potential.

Cellulase is a collective term that encompasses enzymes which catalyze reactions that participate in the degradation of insoluble cellulose to soluble carbohydrates. In the present study, production of extra cellular cellulases by a filamentous fungus, Aspergillus niger FCBP-02, was studied in solid-state fermentation (SSF) as well as in submerged fermentation (SmF). Trials were conducted to evaluate the effect of mutagenesis by UV irradiation (5-40 min) and ethyl methane sulfonate (EMS) treatment (50-300 microg mL(-1)) to obtain hyper active cellulase enzyme producers among the potential strains. The enzyme activity assays of parental and mutant strains clearly revealed significantly higher cellulase activity of mutant A-Ch-5.5 (96 Units mL(-1)), followed by A-UV-5.6 (71 Units mL(-1)) with respect to the wild strain of A. niger FCBP-02 (53.7 Units mL(-1)). The profile of genetic variability among wild and mutant derivatives was scrutinized through RAPD-PCR. The expression pattern of mutants exhibited that the mutants were isogenic variants of the wild type and the out performance of the mutants could be attributed to the change in genetic make up.

Identification and characterization of the most abundant cellulases in stylet secretions from Globodera rostochiensis.

Plant-parasitic cyst nematodes secrete cell wall modifying proteins during their invasion of host plants. In this study, we used a monoclonal antibody to immunopurify and to sequence the N terminus of the most abundant cellulases in stylet secretions of preparasitic juveniles of Globodera rostochiensis. The N-terminal amino acid sequence perfectly matched the sequence of an expressed sequence tag of two nearly identical genes, named Gr-eng3 and Gr-eng4, which show relatively low similarity with the previously identified Gr-eng1 and Gr-eng2 (i.e., 62% similarity and 42% identity). The recombinantly produced proteins from Gr-eng3 and Gr-eng4 demonstrated specific activity on carboxymethylcellulose, indicating that these genes encode active cellulases. To date, the cellulases in cyst nematodes are comprised of three possible domain structure variants with different types of ancillary domains at the C terminus of the glycosyl hydrolase family 5 (GHF5) domain. We used Bayesian inference to show that the phylogeny of the GHF5 domain based on currently available data suggest that the extant nematode cellulases arose through reshuffling of the GHF5 domain with different types of ancillary domains as relatively independent units. Knocking-down Gr-eng3 and Gr-eng4 using RNA interference resulted in a reduction of nematode infectivity by 57%. Our observations show that the reduced infectivity of the nematodes can be attributed to poor penetration of the host's root system at the onset of parasitism.