Cloning, Expression And Directional Modification Of Cbh1and Glucose-tolerant Bgl1Gene From Aspergillus Niger

Cellulosic biomass is the most abundant renewable resource on earth, whose naturaldegradation represents an important part of the carbon cycle within the biosphere. The completedegradation of the cellulose requires the synergistic action of cellulases, which includeendo-β-glucanase, exo-β-glucanase, and β-glucosidase. In this paper, the gene cbh1fromAspergillus niger encoding exo-β-glucanase was cloned and expressed in Pichia pastoris, and thegene bglI encoding β-glucosidase from A. niger was synthesized and mutated for improvementthe expression and glucose tolerance. The results are as follows:(1) The gene cbh1was cloned from Aspergillus niger NL-1by using PCR. The cbh1consists of1515bp, including three introns. It shared the high similarity with other cbh1genefrom Aspergillus sp, which belonged to family7of the glycoside hydrolases.(2) Recombinant plasmid pPICZαA-cbh1was successfully constructed and expressed in thePichia pastoris KM71H. The highest activity of recombinant protein CBH I was125U/L with amolecular weight of approximately60kDa. The optimal activity was at60oC and pH4.0. Therecombinant CBH I was stable over broad pH and relative high temperature.(3) NCBI analysis revealed that there are six rare codons in active site of cbh1gene. Toimprove expression level of recombinant CBH I, rare codons around active site of cbh1werereplaced with optimal codons used in P. pastoris by long-distance inverse PCR. The recombinantplasmid pPICZαA-cbh1m which contains the modified cbh1gene was transformed in P. pastorisKM71H. After methanol induction, the expression level of cbh1m was17%higher than that ofcbh1.(4) Heterologous expression of A. niger cbh1gene in a P. pastoris was tested under theglyceraldehyde-3-phosphate dehydrogenase promoter. The constitutive expression vectorspGAPZαA-cbh1was constructed and transformed into P. pastoris KM71H strain. The activity ofCBH I was210U/L at28oC for3days under GAP promoter, and the highest specific activity was5494U/g.(5) To improve the expression level of recombinant β-glucosidase in P. pastoris, the bgl1gene was designed and synthesized based on the synonymous codon bias of P. pastoris. Theactivity of the recombinant BGL I reached to41.21U/mL, which was1.5higher than that ofunoptimized bgl1gene. The activity of the BGL I reached to53.2U/mL by constructingmulti-copy gene. (6) Flavone compounds and essential oil compounds of Ginkgo tea were extracted andhydrolyzed by β-glucosidase respectively. HPLC showed that part of flavonol glycosidecompounds were hydrolyzed to flavone aglycone. And GC-MS proved that some mellow aromacomponents were relased.(7) In order to improving glucose tolerance of β-glucosidase, three methods wereimplemented.1) By replacing AOX1promoter with GAP promoter, the Ki value of recombinantBGL I for glucose was82.08mM, which was3higher than before.2) By sequence analysis,eleven sites of BGL I related to glucose tolerance were found and site-directed mutated. Theresult showed that none of them were positive effects except the713site.3) A. niger wasdomesticated to get a highly glucose-tolerant strain. Blasted the bgl1gene from this highlyglucose-tolerant strain and initial A. niger, three sites (1,522and685) had mutated. Eightmutation methods were designed and implemented. By now, those methods were completed.