Abstract
BackgroundsThe fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II.ResultsThe transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively.ConclusionsThis work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters.
Highlights
Since cellobiohydrolase I (CBHI) is encoded by single copy of cbh1, the cbh1 promoter is considered to be strong and has been used to produce various kinds of homologous or heterologous proteins [1,7,8,9,10]
As previously described, the expression of these genes in T. reesei might still be affected by glucose concentration [19]. 13 critical genes that participate in glucose metabolism according to Chambergo et al [19] were selected and their transcriptional profiles were quantitatively analyzed by using reverse transcription (RT)-qPCR
These genes include those encoding glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, enolase, alcohol dehydrogenase, triose phosphate isomerase, aldolase, pyruvate kinase, citrate synthase, αketoglutarate dehydrogenase, aldehyde dehydrogenase I, aldehyde dehydrogenase II, pyruvate dehydrogenase, and glucokinase, which participate in glycolysis and the tricarboxylic acid cycle
Summary
Plasmids, and cultivation conditions Escherichia coli (E. coli) Top10F’ (Invitrogen, USA) was used for plasmid construction and maintenance. The E. coli strain was cultivated in LB medium, in which ampicillin (100 μg/ml, Invitrogen) was supplemented when necessary. The T. reesei strain was maintained on potato dextrose agar (PDA), and for liquid cultivation, it was grown in Mandels medium that contained an appropriate concentration of glucose [31]. The recombinant T. reesei strains were selected on PDA agar supplemented with hygromycin B (100 μg/ml), and for recombinant xylanase production, the strains were cultivated in modified Mandels medium supplemented with 7% glucose, 5% soybean powder, and 1% peptone. Plasmid pAN7-1 which contained the hygromycin B resistant cassette was used as an assisting plasmid for the transformation of T. reesei [32]
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