Int. J. Biol. Sci. 2016, Vol. 12 156 Ivyspring International Publisher International Journal of Biological Sciences 2016; 12(2): 156-171. doi: 10.7150/ijbs.13537 Review Recent Developments in Using Advanced Sequencing Technologies for the Genomic Studies of Lignin and Cellulose Degrading Microorganisms Ayyappa kumar Sista Kameshwar, Wensheng Qin Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada. Corresponding author: Email: [email protected], Tel: 807-343-8467 © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions. Received: 2015.08.12; Accepted: 2015.11.03; Published: 2016.01.01 Abstract 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 com- munity 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 se- quencing 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. Key words: Lignocellulose, Fungi, Bacteria, 16s rRNA, Inter Transcribed Spacer, Biodegradation Introduction Drastic change in the climatic conditions and organic polymer on the earth, composed of numerous decrease in the fossil fuel reserves are the main mo- glucose units linked in β (14) linkages, occurring in tives for the extensive research being conducted in the both crystalline and amorphous forms. Cellulose is field of bio refining industry. The abundance and present in plant cell walls, stems, straw, stalks and availability of lignocellulosic biomass makes the cel- other woody portion of the plant, it alone constitutes lulosic ethanol as a significant and immediate alter- to 40-50% of dry weight of plant material [2]. ii) native for conventional fuels. Hemicellulose is a composite compound with a mix- ture of pentoses, hexoses and sugar acids [3-6]. Hem- Lignocellulose - a potential source for future icellulose constitutes a significant component in cell energy needs wall connecting cellulose and lignin layers, thus to- Plant cells are protected with three layers known tally imparting strength and rigidity to the whole as middle lamella, primary and secondary cell walls lignocellulose network[6]. Hemicellulose is also which are made up of carbohydrates (cellulose, hem- studied for its derivative known as xylose, which is icellulose and pectin) and lignin [1] [Figure 1] i) Cel- mainly used for the production of xylitol a valuable lulose is a plant polysaccharide and highly abundant sucrose substituent. iii) Lignin is a complex organic http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 157 biopolymer mainly present in plants in close associa- versatile peroxidases and laccases which are collec- tion with carbohydrates like cellulose and hemicellu- tively involved in the breakdown and utilization of lose, its main activity is to provide plants with struc- lignocellulosic biomass [15]. The property, structure tural stability, impermeability and resistance towards and function of fungal laccases have been studied and microbial attack [3]. It is made up of three phe- reported earlier [16, 17]. Compared to its counterparts nylpropanoid units guaiacyl (G), p-hydroxyphenyl bacterial enzymology for lignin degradation was not (H) and syringyl alcohols (S) also called as monolig- well understood. According to Ramachandran et.al nols, inter linked by several carbon-carbon linkages (2000), Streptococcus viridosporus T7A, a lignin de- [7, 8]. It is hard to effectively use cellulose without grading bacterium produces several extracellular pe- breaking and separating lignin efficiently. Extensive roxidases which are similar to the peroxidases pro- research has been carried out for separating lignin by duced by fungi [18]. Recent advancements in the ge- using chemical, physical and biological methods [9, nome sequencing studies reveal that laccases are also 10]. widespread in several bacterial strains [19]. However the bacterial laccases were not well characterized and Microorganisms as possible degraders of lig- understood as fungal laccases [18-20]. Several molec- nocellulose ular methods are applied in the modern microbiology Fungi are greatly known for their ability to de- to identify and classify the microorganisms such as polymerize lignocellulosic biomass compared to bac- bacteria and fungi. In order to find the molecular teria. Earlier studies convey that several fungal spe- phylogeny of bacteria the 16s rRNA genes are used, cies like Trichoderma reesei, Phanerochaete chrysospori- because this part of DNA was undisturbed and con- um, Fomitopsis palustris, Orpinomyces sp etc, were served during the evolution. Similarly, Inter Tran- found to degrade cellulose by inhabiting the gastro- scribed Spacer (ITS) DNA is used for finding the mo- intestinal tracts of several ruminating animals [11, 12]. lecular ecology of different fungal strains. Wide re- Fungi produce variety of intracellular and extracellu- search has been carried out to find potential lignocel- lar enzymes such as cellulases and hemicellulases [11, lulosic biomass degrading microorganisms [Figure 2]. 13, 14] lignin peroxidases, manganese peroxidases, Figure 1: Schematic representation of plant cell wall components having high commercial importance a) Cellulose b) Hemicellulose c) Cellobiose d) xylose e) Lignin. http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 158 Figure 2: Represents the potential groups of bacteria and fungi, capable of degrading plant cell wall components (cellulose, hemicellulose and lignin). Compiled from [7, 10, 21-23] ject DNA. Maxam Gilbert method mainly have two Genome sequencing studies for the identifica- disadvantages: a) It requires high amounts of quality tion and characterization of microorganisms DNA b) the process cannot be automated. Due to Genome sequencing is a revolutionary method these drawbacks this method was supersede by to analyze the DNA sequence of an organism effi- Sanger’s method of sequencing. ciently. The era of sequencing can be divided into Next Generation Sequencing (NGS): It mostly three successive generations such as: refers to those high throughput technologies which First Generation Sequencing: Sanger sequenc- facilitates parallel sequencing of DNA templates with ing and Maxam Gilbert sequencing comes under the millions of base pairs. NGS can be broadly divided as first generation sequencing techniques. Sanger se- second generation sequencing and third generation quencing was invented by Frederick Sanger in the sequencing methods based on their progressive evo- year 1977 based on the principle “Dideoxynucleotide” lution from the Sanger sequencing method. Three or “Chain termination” method. In this method the major companies such as Roche, Illumina and Life DNA strand is sequenced by using modified dNTP technologies can be considered as second generation known as ddNTP (dideoxynucleotide triphosphates) sequencers. Second generation sequencing platforms which lacks 3’OH group. The 3’OH group is required are based on two principles: a) polymerase based for the formation of a phosphodiester bond between clonal replication of single DNA molecules separated two nucleotides, however the absence of it leads to the on a solid matrix either a bead (or) planar surface b) cessation of the DNA strand elongation. The ddNTPs cyclic sequencing chemistries. Based on these princi- are either radioactively or fluorescently labeled for the ples, sequencing companies define their own proto- detection by automated sequencer machines [24]. col. Apart from the above mentioned similarities, all Sanger sequencing was the first successful method sequencing platforms follow same format of front-end developed for DNA sequencing with innumerable library preparation methods such as adding universal applications in biology and medicine [24]. During adapter sequences to the DNA fragment on its ter- 1976-1977, Maxam Gilbert introduced chemical minal ends. The adapter sequences are complemen- method of DNA sequencing which involved the ra- tary to polymerase chain reaction (PCR) primers dioactive labeling of 5’OH group of DNA strand fol- (which are required for the amplification of the li- lowed by base modification using chemicals and later brary) and also to the oligonucleotides adhered to a the extracted DNA is subjected to electrophoresis and solid support