Glucose-induced secretion of Trichoderma reesei xylanases.
To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.
Kurzatkowski
W
W
National Institute of Hygiene, Warsaw, Poland.
Törrönen
A
A
Filipek
J
J
Mach
R L
RL
Herzog
P
P
Sowka
S
S
Kubicek
C P
CP
D001483:Base Sequence; D005798:Genes, Bacterial; D005947:Glucose / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014242:Trichoderma / Q000201:enzymology*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D005947:Glucose