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Applied and Environmental Microbiology, May 2007, p. 3215-3224, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02967-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Increased Production of Xylanase by Expression of a Truncated Version of the xyn11A Gene from Nonomuraea flexuosa in Trichoderma reesei{triangledown}

Marja Paloheimo,* Arja Mäntylä,{dagger} Jarno Kallio, Terhi Puranen, and Pirkko Suominen{ddagger}

Roal Oy, P.O. Box 57, FIN-05201 Rajamäki, Finland

Received 21 December 2006/ Accepted 13 March 2007

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter–1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.

* Corresponding author. Mailing address: Roal Oy, P.O. Box 57, FIN-05201 Rajamäki, Finland. Phone: 358-9-29042128. Fax: 358-9-29042112. E-mail: marja.paloheimo{at}roal.fi

{triangledown} Published ahead of print on 23 March 2007.

{dagger} Present address: Borenius & Co., Tallberginkatu 2A, FIN-00180 Helsinki, Finland.

{ddagger} Present address: Nature Works LLC, 15305 Minnetonka Blvd., Minnetonka, MN 55345.

Applied and Environmental Microbiology, May 2007, p. 3215-3224, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02967-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Nakari-Setala, T., Paloheimo, M., Kallio, J., Vehmaanpera, J., Penttila, M., Saloheimo, M. (2009). Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein Production. Appl. Environ. Microbiol. 75: 4853-4860 [Abstract] [Full Text]  


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