Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts.
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.
Bergquist
P L
PL
Department of Biological Sciences, and Research Institute for Biotechnology, Macquarie University, Sydney, New South Wales 2109, Australia. peter.bergquist@mq.edu.au
Te'o
V S J
VS
Gibbs
M D
MD
Curach
N C
NC
Nevalainen
K M H
KM
D001671:Biochemistry / Q000379:methods; D001709:Biotechnology / Q000379:methods; D003062:Codon; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry*; D006031:Glycosylation; D006358:Hot Temperature; D007716:Kluyveromyces / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020543:Proteome; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry*; D013997:Time Factors; D014158:Transcription, Genetic; D014242:Trichoderma / Q000378:metabolism*; D001617:beta-Glucosidase / Q000378:metabolism
D003062:Codon; D004798:Enzymes; D020543:Proteome; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase