Genome and transcriptome analysis of rock-dissolving Pseudomonas sp. NLX-4 strain.

Microbial weathering processes can significantly promote soil properties and reduce rock-to-soil ratio. Some soil-inhabiting bacteria exhibit efficient rock-dissolution abilities by releasing organic acids and other chemical elements from the silicate rocks. However, our understanding of the molecular mechanisms involved during bacterial rock-dissolution is still limited. In this study, we performed silicate rock-dissolution experiments on a Pseudomonas sp. NLX-4 strain isolated from an over-exploited mining site. The results revealed that Pseudomonas sp. NLX-4 strain efficiently accelerates the dissolution of silicate rocks by secreting amino acids, exopolysaccharides, and organic acids. Through employing genome and transcriptome sequencing (RNA-seq), we identified the major regulatory genes. Specifically, 15 differentially expressed genes (DEGs) encoding for siderophore transport, EPS and amino acids synthesis, organic acids metabolism, and bacterial resistance to adverse environmental conditions were highly up-regulated in silicate rock cultures of NLX-4 strain. Our study reports a potential bacterial based approach for improving the ecological restoration of over-exploited rock mining sites.

Effect of KNO3 on Lipid Synthesis and CaCO3 Accumulation in Pleurochrysis dentata Coccoliths with a Special Focus on Morphological Characters of Coccolithophores.

Pleurochrysis genus algae are widely distributed in ocean waters. Pleurochrysis sp. algae are popularly known for its coccolithophores. Calcium carbonate (CaCO3) shells are major components of the coccolithophore, and they are key absorbers of carbondioxide. In this study, we have reported the effects of potassium nitrate (KNO3) concentration on calcium accumulation and total lipid, carbohydrate and protein contents of Pleurochrysis dentata. Results obtained from complexometric titration and scanning electron microscopy analysis showed higher rates of CaCO3 accumulation on Pleurochrysis dentata cell surface. We have also observed that overall cell size of Pleurochrysis dentata reached maximum when it was cultured at 0.75 mmol L-1 of KNO3. During 10 days of Pleurochrysis dentata culture total lipids and carbohydrate contents decreased, with slightly increased protein content. Results obtained from Fourier-Transform Infrared Spectroscopy (FTIR) also reported an increase in protein and decrease in lipids and carbohydrate contents, respectively. Similarly, Pleurochrysis dentata cultured at 1 mmol L-1 concentration of KNO3 exhibited the lowest carbohydrate (21.08%) and highest protein (32.87%) contents. Interestingly, Pleurochrysis dentata cultured without KNO3 exhibited 33.61% of total lipid content which reduced to a total lipid content of 13.67% when cultured at 1 mmol L-1 concentration of KNO3. Thus, culture medium containing higher than 1 mmol L-1 of KNO3 could inhibit the cell size of Pleurochrysis dentata and CaCO3 accumulation in shells but it could promote its cell growth. For the first time we have reported a relatively complete coccolith structure devoid of its protoplast. In this study, we have also described about the special planar structure of Pleurochrysis dentata CaCO3 shells present on its inner tube of the R unit and parallel to the outer tube of the V unit which we named it as "doornail structure". We believe that this doornail structure provides structural stability and support to the developing coccoliths in Pleurochrysis dentata. Also, we have discussed about the "double-disc" structure of coccoliths which are closely arranged and interlocked with each other. The double-disc structure ensures fixation of each coccolith and objecting its free horizontal movement and helps in attaining a complementary coccolith structure.

Metadata Analysis Approaches for Understanding and Improving the Functional Involvement of Rumen Microbial Consortium in Digestion and Metabolism of Plant Biomass.

Rumen is one of the most complex gastro-intestinal system in ruminating animals. With bountiful of microorganisms supporting in breakdown and consumption of minerals and nutrients from the complex plant biomass. It is predicted that a table spoon of ruminal fluid can reside up to 150 billion microorganisms including various species of bacteria, fungi and protozoa. Several studies in the past have extensively explained about the structural and functional physiology of the rumen. Studies based on rumen and its microbiota has increased significantly in the last decade to understand and reveal applications of the rumen microbiota in food processing, pharmaceutical, biofuel and biorefining industries. Recent high-throughput meta-genomic and proteomic studies have revealed humongous information on rumen microbial diversity. In this study, we have extensively reviewed and reported present-day's progress in understanding the rumen microbial diversity. As of today, NCBI resides about 821,870 records based on rumen with approximately 889 genome sequencing studies. We have retrieved all the rumen-based records from NCBI and extensively catalogued the rumen microbial diversity and the corresponding genomic and proteomic studies respectively. Also, we have provided a brief inventory of metadata analysis software packages and reviewed the metadata analysis approaches for understanding the functional involvement of these microorganisms. Knowing and understanding the present progress on rumen microbiota and performing metadata analysis studies will significantly benefit the researchers in identifying the molecular mechanisms involved in plant biomass degradation. These studies are also necessary for developing highly efficient microorganisms and enzyme mixtures for enhancing the benefits of cattle-feedstock and biofuel industries.

Isolation and Screening of Cellulose-Degrading Microorganisms from Different Ecological Niches.

Increased interest in developing cellulose-based ethanol over the last few years was the main reason behind inflated research to find cellulose-degrading microorganisms. Several methods have been developed in the past for efficient isolation and characterization of cellulolytic microorganisms. However, it is critical to choose a specific method from a list of qualitative methods for the characterization of cellulose degrading microorganisms. In this chapter, we have extensively listed various qualitative methods used for the isolation and characterization of the cellulolytic microorganisms isolated from different ecological niches such as soil, decaying wood, gut, and rumen.

Purification and Characterization of the Total Cellulase Activities (TCA) of Cellulolytic Microorganisms.

Cellulose is the earth's most abundant plant polysaccharide containing a large array of glucose units linked through ß (1 â†’ 4) linkages by existing in both crystalline and amorphous forms. Cellulose is widely distributed in plants, constituting up to 40-50% overall dry weight of the plant biomass. Majorly, microorganisms secrete three types of enzymes such as endoglucanases, exoglucanases, and beta-glucosidase for the hydrolysis of cellulose, contributing to the total cellulase activity. Industrially, the cellulolytic microorganisms are assessed based on their total cellulolytic activities. Similarly, total cellulase activity can also be used for the isolation and characterization of the cellulolytic microorganisms. In this chapter, we have specifically discussed about the methods used for the purification and characterization of the total cellulase activities of the microorganisms such as filter paper assay and cellulase zymogram assay. Our present chapter can be used as primer for characterizing cellulolytic abilities of cellulose-degrading microorganisms.

Genome Wide Analysis Reveals the Extrinsic Cellulolytic and Biohydrogen Generating Abilities of Neocallimastigomycota Fungi.

Ruminating animals, especially cattle lack the carbohydrate active enzyme encoding genes which are required for the degradation of the glycosidic linkages of plant cell wall carbohydrates (such as cellulose, hemicellulose, lignin and pectin). Thus, ruminating animals are completely dependent on the microorganisms (anaerobic bacteria and fungi, methanogenic archaea and protozoa) residing in their rumen (hindgut). In this study, we have retrieved and analyzed the complete genome wide annotations of the Neocallimastigomycota division fungi such as Anaeromyces robustus, Neocallismatix californiae, Orpinomyces sp, Piromyces finnis, Piromyces sp E2. We have retrieved the InterPro, CAZy, KOG, KEGG, SM Clusters and MEROPS genome level data of these anaerobic fungi from JGI-MycoCosm database. Results obtained in our study reveals that, the genomes of anaerobic fungi completely lack genes encoding for lignin degrading auxiliary activity enzymes. Contrastingly, these fungi outnumbered other fungi by having highest number of CAZyme encoding genes. The genes encoding for dockerins and carbohydrate binding modules exaggerated other CAZymes which are involved in the structure and functioning of cellulosomes. Presence of cellulosomes and higher number of carbohydrate transport and metabolism genes also endorses the plant cell wall carbohydrate degrading abilities of these fungi. We also reported the tentative total cellulolytic, hemicellulolytic and pectinolytic abilities. And we have explicitly reported the genes, enzymes and the mechanisms involved in structure and functioning of the cellulosomes and hydrogenosomes. Our present work reveals the genomic machinery underlying the extrinsic plant cell wall degrading abilities of the anaerobic fungi. Results obtained in our study can be significantly applied in improving the gut health of cattle and especially in the fields of biofuel, biorefining and bioremediation-based industries.

Molecular Networks of Postia placenta Involved in Degradation of Lignocellulosic Biomass Revealed from Metadata Analysis of Open Access Gene Expression Data.

To understand the common gene expression patterns employed by P. placenta during lignocellulose degradation, we have retrieved genome wide transcriptome datasets from NCBI GEO database and analyzed using customized analysis pipeline. We have retrieved the top differentially expressed genes and compared the common significant genes among two different growth conditions. Genes encoding for cellulolytic (GH1, GH3, GH5, GH12, GH16, GH45) and hemicellulolytic (GH10, GH27, GH31, GH35, GH47, GH51, GH55, GH78, GH95) glycoside hydrolase classes were commonly up regulated among all the datasets. Fenton's reaction enzymes (iron homeostasis, reduction, hydrogen peroxide generation) were significantly expressed among all the datasets under lignocellulolytic conditions. Due to the evolutionary loss of genes coding for various lignocellulolytic enzymes (including several cellulases), P. placenta employs hemicellulolytic glycoside hydrolases and Fenton's reactions for the rapid depolymerization of plant cell wall components. Different classes of enzymes involved in aromatic compound degradation, stress responsive and detoxification mechanisms (cytochrome P450 monoxygenases) were found highly expressed in complex plant biomass substrates. We have reported the genome wide expression patterns of genes coding for information, storage and processing (KOG), tentative and predicted molecular networks involved in cellulose, hemicellulose degradation and list of significant protein-ID's commonly expressed among different lignocellulolytic growth conditions.

Comparative modeling and molecular docking analysis of white, brown and soft rot fungal laccases using lignin model compounds for understanding the structural and functional properties of laccases.

Extrinsic catalytic properties of laccase enable it to oxidize a wide range of aromatic (phenolic and non-phenolic) compounds which makes it commercially an important enzyme. In this study, we have extensively compared and analyzed the physico-chemical, structural and functional properties of white, brown and soft rot fungal laccases using standard protein analysis software. We have computationally predicted the three-dimensional comparative models of these laccases and later performed the molecular docking studies using the lignin model compounds. We also report a customizable rapid and reliable protein modelling and docking pipeline for developing structurally and functionally stable protein structures. We have observed that soft rot fungal laccases exhibited comparatively higher structural variation (higher random coil) when compared to brown and white rot fungal laccases. White and brown rot fungal laccase sequences exhibited higher similarity for conserved domains of Trametes versicolor laccase, whereas soft rot fungal laccases shared higher similarity towards conserved domains of Melanocarpus albomyces laccase. Results obtained from molecular docking studies showed that aminoacids PRO, PHE, LEU, LYS and GLN were commonly found to interact with the ligands. We have also observed that white and brown rot fungal laccases showed similar docking patterns (topologically monomer, dimer and trimer bind at same pocket location and tetramer binds at another pocket location) when compared to soft rot fungal laccases. Finally, the binding efficiencies of white and brown rot fungal laccases with lignin model compounds were higher compared to the soft rot fungi. These findings can be further applied in developing genetically efficient laccases which can be applied in growing biofuel and bioremediation industries.

Gene expression metadata analysis reveals molecular mechanisms employed by Phanerochaete chrysosporium during lignin degradation and detoxification of plant extractives.

Lignin, most complex and abundant biopolymer on the earth's surface, attains its stability from intricate polyphenolic units and non-phenolic bonds, making it difficult to depolymerize or separate from other units of biomass. Eccentric lignin degrading ability and availability of annotated genome make Phanerochaete chrysosporium ideal for studying lignin degrading mechanisms. Decoding and understanding the molecular mechanisms underlying the process of lignin degradation will significantly aid the progressing biofuel industries and lead to the production of commercially vital platform chemicals. In this study, we have performed a large-scale metadata analysis to understand the common gene expression patterns of P. chrysosporium during lignin degradation. Gene expression datasets were retrieved from NCBI GEO database and analyzed using GEO2R and Bioconductor packages. Commonly expressed statistically significant genes among different datasets were further considered to understand their involvement in lignin degradation and detoxification mechanisms. We have observed three sets of enzymes commonly expressed during ligninolytic conditions which were later classified into primary ligninolytic, aromatic compound-degrading and other necessary enzymes. Similarly, we have observed three sets of genes coding for detoxification and stress-responsive, phase I and phase II metabolic enzymes. Results obtained in this study indicate the coordinated action of enzymes involved in lignin depolymerization and detoxification-stress responses under ligninolytic conditions. We have developed tentative network of genes and enzymes involved in lignin degradation and detoxification mechanisms by P. chrysosporium based on the literature and results obtained in this study. However, ambiguity raised due to higher expression of several uncharacterized proteins necessitates for further proteomic studies in P. chrysosporium.

Metadata Analysis of Phanerochaete chrysosporium Gene Expression Data Identified Common CAZymes Encoding Gene Expression Profiles Involved in Cellulose and Hemicellulose Degradation.

In literature, extensive studies have been conducted on popular wood degrading white rot fungus, Phanerochaete chrysosporium about its lignin degrading mechanisms compared to the cellulose and hemicellulose degrading abilities. This study delineates cellulose and hemicellulose degrading mechanisms through large scale metadata analysis of P. chrysosporium gene expression data (retrieved from NCBI GEO) to understand the common expression patterns of differentially expressed genes when cultured on different growth substrates. Genes encoding glycoside hydrolase classes commonly expressed during breakdown of cellulose such as GH-5,6,7,9,44,45,48 and hemicellulose are GH-2,8,10,11,26,30,43,47 were found to be highly expressed among varied growth conditions including simple customized and complex natural plant biomass growth mediums. Genes encoding carbohydrate esterase class enzymes CE (1,4,8,9,15,16) polysaccharide lyase class enzymes PL-8 and PL-14, and glycosyl transferases classes GT (1,2,4,8,15,20,35,39,48) were differentially expressed in natural plant biomass growth mediums. Based on these results, P. chrysosporium, on natural plant biomass substrates was found to express lignin and hemicellulose degrading enzymes more than cellulolytic enzymes except GH-61 (LPMO) class enzymes, in early stages. It was observed that the fate of P. chrysosporium transcriptome is significantly affected by the wood substrate provided. We believe, the gene expression findings in this study plays crucial role in developing genetically efficient microbe with effective cellulose and hemicellulose degradation abilities.

The ACEII recombinant Trichoderma reesei QM9414 strains with enhanced xylanase production and its applications in production of xylitol from tree barks.

BACKGROUND: ACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes. Apart from ACEII, transcription factors such as XYR1, CRE1, HAP2/3/5 complex and ACEI function in a coordinated pattern for regulating the gene expression of cellulases and hemicellulases. Studies have demonstrated that ACEII gene deletion results in decreased total cellulase and xylanase activities with reduced transcript levels of lignocellulolytic enzymes. RESULTS: In this study, we have successfully transformed the ACEII transcription factor encoding gene in Trichoderma reesei to significantly improve its degrading abilities. Transformation experiments on parental strain T. reesei QM9414 has resulted in five genetically engineered strains T/Ace2-2, T/Ace2-5, T/Ace2-8, T/Ace5-4 and T/Ace10-1. Among which, T/Ace2-2 has exhibited significant increase in enzyme activity by twofolds, when compared to parental strain. The T/Ace2-2 was cultured on growth substrates containing 2% bark supplemented with (a) sugar free + MA medium (b) glucose + MA medium and (c) xylose + MA medium. The bark degradation efficiency of genetically modified T/Ace2-2 strain was assessed by analyzing the xylitol production yield using HPAEC. By 6th day, about 10.52 g/l of xylitol was produced through enzymatic conversion of bark (2% bark + MA + xylose) by the T/Ace2-2 strain and by 7th day the conversion rate was found to be 0.21 g/g. Obtained results confirmed that bark growth medium supplemented with D-xylose has profoundly increased the conversion rate of bark by T/Ace2-2 strain when compared to sugar free and glucose supplemented growth media. Results obtained from scanning electron microscopy has endorsed our current results. Bark samples inoculated with T/Ace2-2 strain has showed large number of degraded cells with clearly visible cavities and fractures, by exposing the microfibrillar interwoven complex. CONCLUSION: We propose a cost effective and ecofriendly method for the degradation of lignocellulosic biomass such as bark to produce xylitol by using genetically modified T. reesei. Efficient conversion rate and production yield obtained in our current study provides a great scope for the xylitol industries, as our method bypasses the pretreatment of bark achieving clean and low-cost xylitol production.

Recent Developments in Using Advanced Sequencing Technologies for the Genomic Studies of Lignin and Cellulose Degrading Microorganisms.

Lignin is a complex polyphenyl aromatic compound which exists in tight associations with cellulose and hemicellulose to form plant primary and secondary cell wall. Lignocellulose is an abundant renewable biomaterial present on the earth. It has gained much attention in the scientific community in recent years because of its potential applications in bio-based industries. Microbial degradation of lignocellulose polymers was well studied in wood decaying fungi. Based on the plant materials they degrade these fungi were classified as white rot, brown rot and soft rot. However, some groups of bacteria belonging to the actinomycetes, α-proteobacteria and ß-proteobacteria were also found to be efficient in degrading lignocellulosic biomass but not well understood unlike the fungi. In this review we focus on recent advancements deployed for finding and understanding the lignocellulose degradation by microorganisms. Conventional molecular methods like sequencing 16s rRNA and Inter Transcribed Spacer (ITS) regions were used for identification and classification of microbes. Recent progression in genomics mainly next generation sequencing technologies made the whole genome sequencing of microbes possible in a great ease. The whole genome sequence studies reveals high quality information about genes and canonical pathways involved in the lignin and other cell wall components degradation.