Ayyappa Kumar Sista Kameshwar

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 β-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.

Lignin Degrading Fungal Enzymes

Lignin is a complex heterophenolic biopolymer present in plant cell walls. It supports plants growth by providing structural integrity, impermeability, protection against pathogenic infection and pest damage. Being the second most abundant polymer on the earth, it plays a major role in carbon recycling. Increases in greenhouse gas emission and decreases in fuel reserves have increased interest all over the world for the production of biofuels from plant biomass. Because of its complex structure, lignin has become one of the major hurdles for biofuels production. Research has been conducted on methods to separate lignin from lignocellulosic biomass by employing chemical, physical or mechanical methods. However, these methods tend to be expensive and require much energy and also pose risks to the environment. The polyphenolic structure of lignin has attracted interest for the production of renewable and commercially valuable platform chemicals, thus studies have been conducted on its degradation by enzymes. This chapter summarizes recent advances in lignin degrading enzymes (Lignin Oxidizing and Lignin Degrading Auxiliary enzymes) produced by wood degrading fungi and bacteria. Structural and functional aspects of lignin degrading and their auxiliary enzymes are covered with a short note being made on genomic studies of lignin degrading fungi.

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.

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.

Qualitative and Quantitative Methods for Isolation and Characterization of Lignin-Modifying Enzymes Secreted by Microorganisms

Lignin is a polyphenolic biopolymer synthesized by plants, for providing strength and rigidity to the plant cellular structure. It is closely associated with other plant polysaccharides in the cell wall such as cellulose and hemicellulose, constituting the most abundant biopolymer on the earth’s surface. However, the complete utilization of it is being explored for the past few years. Various research groups around the world are trying to replace conventional fuels with the second-generation biofuels from lignocellulose. Several physical, chemical, and biological conversion methods have been developed for the separation and utilization of this biomass, as a result of which biological methods for lignocellulose conversion are considered to be cheap and environment-friendly. Microorganisms, especially fungi and bacteria, have been able to degrade the lignocellulose network and convert it to commercially important biofuels by secreting several intra- and extra-cellular enzymes. In the past few years, research has been conducted to isolate efficient lignin-degrading microorganisms, as separation of lignin from cellulosic biomass is considered as a major hurdle in biofuel and pulping industries. In this article, we extensively discuss different small- and large-scale methods developed for the isolation and characterization of lignin-degrading microorganisms. We have also comprehensively discussed about the qualitative and quantitative methods for the identification and characterization of the lignin-degrading and lignin-degrading auxiliary enzymes by comparing different methods based on their efficiency. This review can be used as a primer for understanding and selecting the most efficient method for isolation and characterization of lignin-degrading microorganisms and their enzymes.

Comparative study of genome-wide plant biomass-degrading CAZymes in white rot, brown rot and soft rot fungi

ABSTRACT We have conducted a genome-level comparative study of basidiomycetes wood-rotting fungi (white, brown and soft rot) to understand the total plant biomass (lignin, cellulose, hemicellulose and pectin) -degrading abilities. We have retrieved the genome-level annotations of well-known 14 white rot fungi, 15 brown rot fungi and 13 soft rot fungi. Based on the previous literature and the annotations obtained from CAZy (carbohydrate-active enzyme) database, we have separated the genome-wide CAZymes of the selected fungi into lignin-, cellulose-, hemicellulose- and pectin-degrading enzymes. Results obtained in our study reveal that white rot fungi, especially Pleurotus eryngii and Pleurotus ostreatus potentially possess high ligninolytic ability, and soft rot fungi, especially Botryosphaeria dothidea and Fusarium oxysporum sp., potentially possess high cellulolytic, hemicellulolytic and pectinolytic abilities. The total number of genes encoding for cytochrome P450 monooxygenases and metabolic processes were high in soft and white rot fungi. We have tentatively calculated the overall lignocellulolytic abilities among the selected wood-rotting fungi which suggests that white rot fungi possess higher lignin and soft rot fungi potentially possess higher cellulolytic, hemicellulolytic and pectinolytic abilities. This approach can be applied industrially to efficiently find lignocellulolytic and aromatic compound-degrading fungi based on their genomic abilities.

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.

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

ABSTRACT Acetyl and methyl esterifications are two major naturally found substitutions in the plant cell-wall polysaccharides. The non-cellulosic plant cell-wall polysaccharides such as pectin and hemicellulose are differentially esterified by the O-acetyl and methyl groups to cease the action of various hydrolytic enzymes secreted by different fungi and bacterial species. Thus, microorganisms have emerged with a special class of enzymes known as carbohydrate esterases (CE). The CE catalyse O-de, N-deacetylation of acetylated saccharide residues (esters or amides, where sugars play the role of alcohol/amine/acid). Carbohydrate active enzyme (CAZy) database has classified CE into 16 classes, of which hemicellulose deacetylating CE were grouped into eight classes (CE-1 to CE-7 and CE-16). Various plant biomass degrading fungi and bacteria secretes acetyl xylan esterases (AcXE); however, these enzymes exhibit varied substrate specificities. AcXE and xylanases-coupled pretreatment methods exhibit significant applications, such as enhancing animal feedstock, baking industry, production of food additives, paper and pulp, xylitol production and biorefinery-based industries, respectively. Thus, understanding the structural and functional properties of acetyl xylan esterase will significantly aid in developing the efficient AcXE with wide range of industrial applications.

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.

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. Fentons 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 Fentons 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-IDs commonly expressed among different lignocellulolytic growth conditions.