Analysis of Functional Genes in Carbohydrate Metabolic Pathway of Anaerobic Rumen Fungus Neocallimastix Frontalis PMA02
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1555 Asian-Aust. J. Anim. Sci. Vol. 22, No. 11 : 1555 - 1565 November 2009 www.ajas.info Analysis of Functional Genes in Carbohydrate Metabolic Pathway of Anaerobic Rumen Fungus Neocallimastix frontalis PMA02 Mi Kown* 2, Jaeyong Song1, 3, Jong K. Ha3, Hong-Seog Park4 and Jongsoo Chang1, * 1 Department of Agricultural Science, Korea National Open University, Korea ABSTRACT : Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastixfrontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five「-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis. (Key Words : Neocallimastix frontalis, EST, ^-Glucosidase, Cellulase, Hemicellulase, Carbohydrate Metabolism) INTRODUCTION carbohydrate degrading enzymes including endoglucanase (Barichieich and Calza, 1990), exoglucanase (Mountfort Ruminant animals use fibrous plant materials as feed by and Asher, 1985), xylanase (Teunissen et al., 1993), and p- degrading insoluble polysaccharides during microbial glucosidase (Li and Calza, 1991). For this reason, the fermentation. Bacteria, fungi and protozoa are the main anaerobic rumen fungi have been regarded as good genetic microorganisms in the rumen ecosystem. After the first resources for enzyme production which might be useful for identification of the anaerobic fungus Neocallimastix animal production, bio-energy production, bio-remediation frontalis (Orpin, 1975), 6 genera including Anaeromyces, and other industrial purposes. However, due to the limited Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces and number of research groups for anaerobic fungi in the world, Piromyces were isolated and identified from the gut of the available genetic information about carbohydrate herbivorous animals. The anaerobic fungi contribute mainly metabolism for anaerobic fungi is limited. In the public data to fiber digestion in the rumen with their effective base, 1,430 nucleotide sequences including 384 core cellulolytic enzymes as well as physical penetration of nucleotides, 996 EST (expression sequence tag) and 247 fungal rhizoid into the fiber matrix (Ho and Abdullah, protein sequences from the family Neocallimastigaceae are 1988). The anaerobic fungi secrete various kinds of currently available (http://www.ncbi.nlm.nih.gov/). Among 247 protein sequences, 70 glucose-hydrolyzing enzyme * Corresponding Author: Jongsoo Chang. Tel: +82-2-3668-4636, sequences and 33 xylose-hydrolyzing enzyme sequences are Fax: +82-2-3668-4187, E-mail: [email protected] currently available. For N. frontalis, 68 nucleotide 2 Department of Forest Products, Kookmin University, Korea. sequences and 44 protein sequences for basic carbohydrate 3 Department of Food and animal biotechnology, Seoul National metabolism, respiration and house-keeping proteins are University, Korea. available in the public data base (http://www.ncbi.nlm.nih.gov/). 4 Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Korea. Among 44 available protein sequences from N. frontalis, Received July 4, 2008; Accepted April 27, 2009 only 4 sequences encoding glucose-hydrolyzing enzyme are 1556 Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 available in the public data base (http://www.ncbi.nlm.nih.gov/). Technologies Inc, USA) and agarose gel electrophoresis, When a nucleotide sequence encoding cellobiohydrolase respectively. The mRNAs were further purified from total (celB, AY328465.1) was searched using the BLAST RNAs using Absolute mRNA Purification Kit (Strtagene, algorithm, more than 8 nucleotide sequences encoding USA) according to the manufacturer’s instruction. The cellobiohydrolase from Orpinomyces, Piromyces and other cDNAs were synthesized with 5 卩g of mRNA using ZAP Neocallimastix species were matched with more than 79% Express® cDNA Synthesis Kit (Startagene, USA). In detail, of identity and 0.0 E-value. The genera Neocallimastix, the mixture of 5 卩l of 10X first-strand buffers, 3 卩l of first- Orpinomyces and Piromyces are members of Family strand methyl nucleotide mixture, 2 卩l of linker-primer, 1 卩l Neocallimastigaceae and they are very close in genetic of RNase block Ribonuclease Inhibitor (40 U/卩l), 5 卩g of relation and the genes encoding glucose hydrolyzing poly(A) RNA, 25 卩l of RNA, 1.5 MMLV-RT (50 U/jU) enzymes are highly homologous among anaerobic fungi reagent and 11 卩l of diethylpyrocarbonate(DEPC)-treated (Harhangi et al., 2003). In addition, these genes are even distilled water was incubated for 1 h at 42°C for first-strand homologous to those in anaerobic bacteria due to horizontal cDNAs synthesis; 50 卩l of ice cooled first-strand cDNAs gene transfer (Garcia-Vallve et al., 2000). For this reason, there might be a strong possibility that N. frontalis secrete were mixed with 20 卩l of 10X second-strand buffer, 6 卩l of more than 4 glucose hydrolyzing enzymes which have not second-strand dNTP mixture, 111 jl of sterile distilled been detected. water, 2 卩l of E. coli RNase H (1.5 U/jl) and 1 卩l of E. coli Many different experimental techniques can be applied DNA polymerase II (9.0 U/jl) and incubated for 2.5 h at to find functional genes from an organism. One 16°C. Subsequently, second-strand cDNAs were mixed experimental technique, high-throughput expressed with 23 jl of blunting dNTP mix and 2 jl cloned Pfu DNA sequence tag (EST) analysis of complementary DNA polymerase (2.5 U/jl) and incubated for 30 min at 72 °C. (cDNA), provides important information about functional After phenol-chloroform (1:1) and chloroform extraction, genes with reasonable cost (Nagaraj et al., 2006). the precipitated cDNA pellet was washed with 20 jl of 3 M Comparison of homology of multiple genes among multiple sodium acetate and subsequently 400 jl of ethanol mixture organisms during evolution is also available with EST at -20°C. The washed cDNA pellet was dried, suspended analysis (Brinkmann et al., 2005). The purpose of this study with 8 卩l of EcoR I adapter and incubated at 4°C for 30 min. was to isolate functional genes related with carbohydrate for blunting the cDNA termini. For ligation of the EcoRI metabolism from the anaerobic rumen fungus N. frontalis adapters, blunted cDNAs were mixed with 1 jl of 10x using high throughput EST analysis for further research. ligase buffer, 1 jl of 10 mM rATP and 1 jl of T4 DNA ligase(4 U/jl) and incubated overnight at 8°C. The reaction MATERIALS AND METHODS was terminated by heating at 70°C for 30 min. After ligation, the reaction mixture was mixed with 1 l of 10 ligase Strains and culture condition j x Neocallimastix frontalis PMA02 was isolated from the buffer, 2 jl of 10 mM rAMP, 5 jl of distilled water and 2 jl rumen of a Holstein steer using Hungate’s roll tube method of T4 polynucleotide kinase (5 U/jl) and incubated for 30 (Hungate, 1966). Isolated fungus was identified according min at 37°C for phosphorylation. The reaction was to morphological characteristics and rRNA ITS1 sequence inactivated by heating at 70°C for 30 min. (Brookman et al., 2000). The rRNA ITS1 sequence with Phosphorylated cDNAs were mixed with 28 jl of XhoI 680 bp size from N. frontalis PMA02 was homologous to buffer and 3 jl of XhoI (40 U/jl) and incubated for 1.5 h at that from N. frontalis SR4 (Fliegerova et al., 2004; 37°C for digestion and fractionated by molecular size using AY429664) with 99% of identity. The fungal strain was a separose CL-2B gel filtration column. The sizes of cultivated with modified Lowe’s medium (Lowe et al., cDNAs were confirmed using 5% non-denaturing 1985) containing 2% of glucose, cellobiose, and starch acrylamide gel and the cDNA fragments with size over 400 (2:1:1) mixture as carbohydrate source. After incubation at bp were collected. cDNAs were ligated into ZAP 39°C for 72 h under anaerobic condition, fungal cells were Expression vector (Stratagene, USA) according to the harvested and stored at -80°C until use. manufacturer’s instruction. The ligated cDNAs were packed into Gigapack III Gold packing extract (Stratagene, USA) RNA extraction and cDNA library construction and transfected into E. coli XL1-Blue MRF’. After Fungal cells were homogenized under liquid nitrogen incubation for 2 h at 22°C, cell debris was removed and and total RNAs were extracted using TrizolTM reagent phage-containing supernatant was collected. The primary (Invitrogen, USA) according to the manufacturer’s library was titrated with SM buffer to 107 pfu lamda phage instruction. The quantity and quality of extracted RNAs concentration and stored at 4°C. The mixture of 108 pfu were determined using a spectrophotometer (Nanodrop XL1-Blue MRF cell and 109 pfu ExAssist helper phage was Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 1557 packed into lamda phage.