Cloning And Analysis Of Function Gene For Cellobiose Metabolism From Pichia Stipitis

Plant fiber is the most widely, the most cheap resources on the earth, which is“inexhaustible in supply and always available for use”, and it is one of the most usefulresources. As the shortage of non-renewable resources such as oil, plant fiber is more andmore attention by researchers. Fuel ethanol production with plant fiber has become the newtrend of energy use.Cellobiase activity is very low in the process of cellulose metabolism, because ofcellulase is insufficient which caused the accumulation of cellobiose. The accumulation ofcellobiose became strong feedback inhibition to cellulose enzyme catalysis, so the research ofcellobiose metabolism is very urgent. As a microbe could hydrolyzed plant fiber, Pichiastipitis was proved having good transport and utilization ability of the fibers. Thus Pichiastipitis became one of the most hot strain of industrial application foreground. However,Pichia stipitis metabolism netwok of cellobiose is still not clear.In order to find cellobiase gene of Pichia stipitis, Pichia stipitis genomic library wasbuilded as a platform of cellobiose metabolism netwok research in our study. The librarycovered1.08times of the15441179bp genomic DNA of the strain, and contained3000clones with the insertion sizes ranging from0.5to8.0Kb. It has a probability of97%forscreening a random gene form this library. The ratio of insert fragment existion inrecombinant plasmids was70%-80%. A medium called YNBC was designed according tothe cellobiase substrate specificity, which cellobiose is the sole carbon source. The libraryplasmids were extracted and transformed into Saccharomyces cerevisiae W303-1A by LiAcmethod. At last, a1.82Kb base sequence named BGL-X of P. stipitis was firstly characterizedby means of screening the transformants on cellobiose plates. There were the fragment1with1485bp probably from Pichia stipitis mitochondrial genome, the fragment2with193bpfrom Pichia stipitis chromosome5and the fragment3with143bp from Pichia stipitischromosome4by BLAST on the NCBI.In order to confirm which base sequence are useful, seven reconstruction plasmids,pYX212-X123, pYX212-X12, pYX212-X23, pYX212-X13, pYX212-X1, pYX212-X2,pYX212-X3, were constructed. Then the seven reconstruction plasmids were transformed intoS. cererisiae W303-1A by LiAc method respectively. Cellobiase activity was detected bypNPG. The transformants with pYX212-X123and pYX212-X12had cellobiase activity. Itmeams that the fragment2plays a very important role in cellobiose metabolism. Thefragment2was searched by BLAST in NCBI. We found that it was located in a3877bpuncommented sequence. And then, a1020bp open reading frame contained fragment2,named BGL, was predicted by ORF Finder in NCBI. In order to further verify the function ofBGL gene, recombinant strains Escherichia coli BL21(PT7-BGL) and Saccharomycescerevisiae W303-1A (PTP1-BGL) were structured. Cellobiase activity was detected by pNPG.The result displayed that BGL expressed cellobiase in intracellular. The gene sequence of BGLwas submitted to NCBI, and accession number is KC677697.