L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.
BACKGROUND
Trichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the L-methionine repressible MET3 gene, encoding ATP sulphurylase.


RESULTS
We show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are L-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including D-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei.


CONCLUSIONS
We describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an L-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.
Austrian Centre of Industrial Biotechnology (ACIB) GmbH c/o Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. robert.bischof@acib.at.
Robert H
RH
Bischof
Austrian Centre of Industrial Biotechnology (ACIB) GmbH c/o Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. jenniferhorejs@gmail.com.|Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. jenniferhorejs@gmail.com.
Jennifer
J
Horejs
Austrian Centre of Industrial Biotechnology (ACIB) GmbH c/o Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. benjaminmetz@gmx.at.|Vogelbusch Biocommodities GmbH, Blechturmgasse 11, 1051, Vienna, Austria. benjaminmetz@gmx.at.
Benjamin
B
Metz
Biotech and Renewables Center, Clariant GmbH, 81477, Munich, Germany. christian.gamauf@clariant.com.
Christian
C
Gamauf
Austrian Centre of Industrial Biotechnology (ACIB) GmbH c/o Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. ckubicek@mail.tuwien.ac.at.|Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. ckubicek@mail.tuwien.ac.at.
Christian P
CP
Kubicek
Austrian Centre of Industrial Biotechnology (ACIB) GmbH c/o Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. bernhard.seiboth@tuwien.ac.at.|Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, Technische Universität Wien, Gumpendorferstraße 1a, 1060, Vienna, Austria. bernhard.seiboth@tuwien.ac.at.
Bernhard
B
Seiboth
D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008715:Methionine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013430:Sulfate Adenylyltransferase / Q000235:genetics*; D014242:Trichoderma / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008715:Methionine; D013430:Sulfate Adenylyltransferase