P. L. Bergquist,1,2
V. S. J. Te’o,1 N.
C. Curach,1 M. D. Gibbs1 and K. M. H. Nevalainen1
1Department of Biological Sciences
Biotechnology
Research Institute
Phone: +61 2 9850 8614
Fax: +61 2 9850 9748
Email: peter.bergquist@mq.edu.au
2Department of Molecular Medicine
& Pathology
University
of
An efficient expression system is the key for the
economically viable bulk production of enzymes for industrial processes. Expression vectors for Kluyveromyces lactis utilise 2m-like plasmid pKD1 replicon and the K. lactis LAC4 promoter. Recombinant proteins in Trichoderma reesei are expressed under the strong cellobiohydrolase
1 (cbh1) promoter as a fusion to the
secretion signal or the mature CBHI protein sequences. We have also reconstructed some of the
thermophile genes according to fungal codon preferences and demonstrated a
dramatic increase in protein production.
We have examined the effect of the expression host on the form and
activity of the heterologous Dictyoglomus
thermophilum xylanase, XynB, produced in different Trichoderma strains. XynB
was produced in multiple forms in these hosts, the pattern depending on whether
all of the potential N-linked glycosylation sites were left intact or
removed. A proteomics approach has been
undertaken to identify powerfully expressed proteins produced by T. reesei in order to identify
condition-specific promoters. One of
these proteins is HEX1, the major protein of the fungal Woronin body that is
predominant under both cellulase-inducing and repressing conditions. The hex1
gene promoter and terminator sequences have been isolated to make an expression
cassette. The promoter function has been
studied in relation to cultivation time and medium composition and appears
promising because of its early onset and extensive production of mRNA.