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.
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.
Li
Xin-Liang
XL
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, United States Department of Agriculture-Agricultural Research Service, 1815 N. University Street, Peoria, IL, USA. lix@ncaur.usda.gov
Skory
Christopher D
CD
Ximenes
Eduardo A
EA
Jordan
Douglas B
DB
Dien
Bruce S
BS
Hughes
Stephen R
SR
Cotta
Michael A
MA
D020856:AT Rich Sequence / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D035901:Hypocrea / Q000235:genetics*; D008969:Molecular Sequence Data; D020085:Neocallimastigales / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D043364:Endo-1,4-beta Xylanases