Evaluation and characterization of Trichoderma reesei cellulase and xylanase promoters.
Comprehensive analyses on promoters of four cellulase and one xylanase genes of Trichoderma reesei were performed expressing a single reporter uidA from Escherichia coli to construct highly functional cellulase-overproducing strains. GUS amount expressed under each promoter correlated entirely with each mRNA amount, suggesting that GUS production was controlled at the transcriptional level. The uidA transcript levels were much lower than the native gene mRNAs, but they were produced in proportion to the mRNA of native cellulase and xylanase genes driven by the same promoters except for the cbh2 promoter. Cellulose-degrading activity and protein amount was reduced in cbh1 and cbh2 disruptant mutants compared to the wild-type T. reesei PC-3-7 and other uidA transformants. The cbh1 disruptant strain was observed to produce more CBH II, EG I, EG III, and xylanases than native PC-3-7 and the other uidA transformants with the same amounts of protein in SDS-PAGE gels. This observation was further analyzed by measuring mRNA levels of cellulase and xylanase genes in the disruptants using quantitative real-time PCR. In the Pcbh1-gus, mRNA levels for cbh2 and egl1 genes were higher than those in native T. reesei PC-3-7 and all other disruptant strains. The cbh2 disruptant strain had the highest amount of cbh1 mRNA among the strains tested. Homologous integration of uidA at the egl1, egl3, and xyn3 loci was also found to cause a slight increased level of cbh1 mRNA, whereas mRNA levels for egl1, egl3, and xyn3 in all the disruptants were similar to those of T. reesei PC-3-7.
Rahman
Zinnia
Z
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2137, Japan.
Shida
Yosuke
Y
Furukawa
Takanori
T
Suzuki
Yota
Y
Okada
Hirofumi
H
Ogasawara
Wataru
W
Morikawa
Yasushi
Y
D044602:Cellulases / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D004274:DNA, Recombinant / Q000032:analysis / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D005798:Genes, Bacterial; D017930:Genes, Reporter; D007218:Industrial Microbiology / Q000379:methods; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
D004274:DNA, Recombinant; D012333:RNA, Messenger; D002482:Cellulose; D044602:Cellulases; D043364:Endo-1,4-beta Xylanases