Evaluation and characterization of Trichoderma reesei cellulase and xylanase promoters

Abstract

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.

Application ofTrichoderma reeseiCellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

“…2, 4) and heterologous gus. 18) We have found that no xyn3 transcripts were detected in T. reesei QM 9414, although the same xyn3 structural gene and 5 0 -upstream region were present. 7,39) We also observed that expression of xyn3 in QM6a was very weak compared to that of the PC-3-7 (unpublished data).…”

“…The GUS amounts expressed under the cbh1, cbh2, egl1, egl3, and xyn3 promoters were, relative to cbh1, 100, 20, 25, 5, and 10%. 18) Because disruption of cbh1, cbh2, and egl1 lowers cellulase activity, we propose that minor promoter egl3 and xyn3 are suitable for homologous recombination, since deletion of egl3 and xyn3 does not affect the cellulase activity. Thus, the egl3 and xyn3 promoters are optimal for the expression y To whom correspondence should be addressed.…”

“…Although these promoters are 10-to 20-fold less active than the cbh1 promoter, they are still 50-to 100-fold stronger than the promoter of the phosphoglycerate kinase 1 gene (pgk1). 18) In T. reesei, one of the major drawbacks in cellulosic biomass degradation is the low level of -glucosidase activity, 19,20) which leads to incomplete conversion of cellobiose to glucose during the hydrolysis of cellulose. To overcome this disadvantage, cellulase preparations of T. reesei must be supplemented with -glucosidase from other sources to increase the rate and extent of cellulose hydrolysis.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Similarly, T. reesei produces cellulase component (endoglucanase and exoglucanase) in high amount with excellent properties such as thermostability and catalytic efficiency but it lacks sufficient amount of β-glucosidase. Researchers therefore have heterologously expressed exogenous β-glucosidase genes in T. reesei under strong promoter in order to enhance its β-glucosidase activity so that cellulose hydrolysis rate was improved considerably [112,187,190,191,192]. Majority of recombinant fungal β-glucosidase belong to glycoside hydrolase family 3.…”

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger

“…However, these promoters are either constitutively active and growth related (e.g., the Aspergillus niger glyceraldehyde-3-phosphate dehydrogenase promoter, PgpdA) or dependent on the carbon or nitrogen source (e.g., A. niger PglaA and PinuE, Aspergillus nidulans PalcA, Neurospora crassa Pqa-2, Ustilago maydis Pcrg1 and Pnar1, and Trichoderma reesei Pcbh1), and some are also leaky (e.g., PinuE and Pqa-2) (7,22,36,37). In search of promoters that are tight, tunable, and metabolism independent, three systems have recently been tested for application in filamentous fungi: the thiamine promoter system (PthiA) in Aspergillus oryzae (40), the human estrogen receptor (hER␣) system in A. nidulans and A. niger (32), and a system based on the Escherichia coli tetracycline resistance operon (Tet) in Aspergillus fumigatus (45).…”