Construction Of The Subtractive CDNA Library Of Volvariella Volvacea. Cloning And Expression Of Some Genes Encoding Polysaccharide Hydrolases

Cultivation of edible mushrooms on cellulosic wastes represents a prime example of thedirect conversion of low-grade residues into a value-added food source and also as a source ofcommercially important metabolites. The edible straw mushroom, V. volvacea, is grown on anindustrial scale in many tropical and subtropical regions and currently ranks fifth among theworld's most important commercially cultivated species. It grows faster at higher temperaturethan other mesophilic filamentous fungi when using straw as substrate. These characteristicsindicate that V. volvacea has a whole series of cellulolytic enzymes which may have betterstability than those from other mesophiles. In this paper we constructed the subtractive cDNAlibrary of V. volvacea V1-1, isolated and cloned genes involved in some polysaccharidedegradation and described their expression in the heterogenous expression systems.1. The strain V. volvacea V1-1 was grown on the optimal inducing medium containing (g/l):0.2% xylan, 0.2% CMC, 0.2% (NH₄)₂SO₄, 0.2% KH₂PO₄, 0.1% MgSO₄, 2.5 mg/L VB1. Theoptimal conditions were pH 6.5 and 150 rpm. We provided a modified method to isolatehigh-quality total RNA from basidomycetes V. volvacea. The purity of RNA was enough to beused in standard and downstream applications such as cDNA synthesis. A rapid andconvenient method for cDNA subtractive library construction was developed. We preparedthe mycelia of V. volvacea under induced conditions and no induced conditions. Two kinds ofmRNA of V. volvacea were extracted. The induced mRNA was reverse-transcripted intosingle-strand cDNA with PowerScript? Reverse Transcriptase. The hybridization betweenthe single-strand cDNA and no induced mRNA was performed. After high stringent washing,purification and concentration, the remained single-strand cDNA were constructed intosubtractive library with SMARTTM cDNA Library Kit. The result revealed that the tilter ofcDNA subtractive library was 2.5×105 pfu/ml. The tilter of amplified cDNA subtractivelibrary was 1.1×109 pfu/ml, the recombinant rate reached 93%, and the length of insertsranged from 0.5 kb to 4.0 kb.2. We for the first time reported the large-scale alternative splicing of xylanase in V.volvacea for introns or exon ambiguities. V. volvacea were likely to need a regulating way toadapt environments of different steps of growth with expression of different kinds andquantities of proteins. It is unknown about the mechanisms underlying alternative splicing ofxylanase in V. volvacea. One of PCR sample was converted by site directed mutagenesis andthe mutant gene could be translated correctly. The mutant gene was transformed into E. colior Pichia pastoris. No xylanase activity was detected in cell disruption of E. coli or P.pastoris. No post-translational modification in E. coli or no correct and complete xylanasegene sequence was probably responsible for no xylanase activity. It needs further research torealize expression of active xylanase.3. A β-glucosidase gene from V. volvacea V1-1 was for the first time expressed in E. coli,and the induced temperature and vector were the key factors affecting the activities of theenzyme expressed in E. coli. The gene product was purified by chromatography showing asingle band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa withsubunits of 97 kDa. The maximum activity was at pH 6.4 and 50?C over the 5 min assay. Thepurified enzyme was stable from pH 5.6 to 8.0, had a half life of 1 h at 45?C, activated byTritonX-100, and sensitive to Cu2+, Hg2+, Zn2+ and SDS. The β-glucosidase was activeagainst p-nitrophenyl-β-D-glucopyranoside (pNPG), cellobiose and p-nitrophenyl-α-D-xylopyranoside. The β-glucosidase had a Km of 0.2 mM and a Vmax of 93 U/mg for pNPG.4. The chitin deacetylase gene (cda) was for the first time cloned from the subtractive cDNAlibrary of V. volvacea V1-1 by a PCR-based strategy using degenerate primers, and theGeneBank accession No. of cda is AY855921. The open reading frame of cda consisted of apossible prepro-sequence of 18 amino acids at the N-terminus and a mature chitin deacetylase.The deduced amino acid sequence of the mature enzyme revealed 39% identity and 47%similarity with cda from Schizophyllum commune. The strain harboring pET-20b-CDAproduced some chitin deacetylase. The chitin deacetylase activities were restored from theinclusion bodies by a simple renaturation step with urea treatment, and the enzyme had aspecific activity of 0.06 unit/mg of protein.