Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.
Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts
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P.L. Bergquist, V.S.J. Te'o, M.D. Gibbs, N.C. Curach, K.M.H. Nevalainen; Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts. Biochem Soc Trans 1 April 2004; 32 (2): 293–297. doi: https://doi.org/10.1042/bst0320293
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