Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei.
The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.
Margolles-Clark
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E
VTT Biotechnology and Food Research, Espoo, Finland.
Hayes
C K
CK
Harman
G E
GE
Penttilä
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M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D014170:Transformation, Genetic; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D003470:Culture Media; D017931:DNA Primers; D004271:DNA, Fungal; D021382:Protein Sorting Signals; D002688:Chitinases