Glucose-induced secretion of Trichoderma reesei xylanases

Abstract

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.

The Promoter Toolbox for Recombinant Gene Expression in Trichoderma reesei

“…The homologous cellobiohydrolase cel7a was overexpressed under P eno1 control in a Δ cel7a strain resulting in suitable product levels (Linger et al, 2015 ). The promoter of pki1 was used for e.g., xylanase production exhibiting low to medium strength (Kurzatkowski et al, 1996 ).…”

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

“…Most of the evaluations of promoter activity in Trichoderma have been done by assessing the expression of genes encoding for xylanases, laccases, and glucoamylases or of reporter genes such as gfp and mCherry [12,17,18,20]. In the case of hydrolytic enzymes, it requires to set up laborious protocols to extract the enzymes and assess their functionality under optimal conditions, whereas in the case of fluorescent proteins the constant use of fluorescence microscopy or fluorometers is required to evaluate promoter activity at specific stage of growth.…”

Novel genetic tools that enable highly pure protein production in Trichoderma reesei

“…Cellobiohydrolase I (CBHI) alone represents about 50–60% of total secreted proteins 7 . While co-expression of the cellulolytic enzymes can be a highly useful for production of cellulolytic enzyme cocktails 7 , these side activities can be undesired or harmful when heterologous proteins are produced in these conditions 19,20 .…”