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Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC-2 in and secretion of the heterologous enzyme by Hypocrea jecorina

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Abstract

The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene (xynA) of the anaerobic fungus Orpinomyces was expressed in Hypocrea jecorina under the control of the cel7A promoter and terminator. No XynA protein was detected in H. jecorina culture supernatants when the original sequence was fused to the H. jecorina cel5A region coding for its signal peptide, carbohydrate-binding module, and hinge. Replacing the xynA (56% AT content) with a synthetic sequence containing lower AT content (39%) supported the extracellular production (150 mg l−1) of the fusion xylanase by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content was related to higher levels of the xylanase-specific mRNA. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed production of the fully processed active xylanase catalytic domain. Both the fusion (40 kDa) and the fully processed (28 kDa) forms displayed enzymatic properties of family 11 xylanases. Both the R and the Kex2-like KR sites were recognized during secretion, resulting in a mixture of two amino termini for the 28-kDa xylanase. The work demonstrated for the first time that glycoside hydrolases derived from anaerobic fungi can be produced by H. jecorina.

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Acknowledgment

The authors thank Patrick M. Kane, Patricia J. O’Bryan, Kristina Glenzinski, Jay Braker, and Joy Danniel for technical support and WIPF for the N-terminal sequencing and LC/MS/MS analyses.

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Correspondence to Xin-Liang Li.

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The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.

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Li, X., Skory, C.D., Ximenes, E.A. et al. Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC-2 in and secretion of the heterologous enzyme by Hypocrea jecorina . Appl Microbiol Biotechnol 74, 1264–1275 (2007). https://doi.org/10.1007/s00253-006-0787-6

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Keywords

  • Orpinomyces
  • Xylanase
  • Trichoderma
  • Hypocrea
  • Hemicellulose