INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS
Location: National Center for Agricultural Utilization Research
Title: 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
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 30, 2006
Publication Date: January 16, 2007
Citation: Li, X., Skory, C.D., Ximenes, E.A., Jordan, D.B., Dien, B.S., Hughes, S.R., Cotta, M.A. 2007. 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. Applied Microbiology and Biotechnology. 74:1264-1275.
Interpretive Summary: Efficient conversion of lignocellulosic biomass to fermentable sugars is recognized as a major bottleneck for the economical production of biofuels and feedstock chemicals from the almost infinite renewable resources. There are three major constitutes, cellulose, hemicellulose, and lignin, commonly found in lignocellulosic biomass. Biological degradation of the three constitutes requires many different enzymes working together in concert. The most needed enzymes are those which tackle the backbone glycosidic chains in cellulose and hemicelluloses. An enzyme required for the hydrolysis of hemicelluloses is a xylanase. Numerous xylanases have been found from microbes, and many of the genes coding for these enzymes have been cloned and sequenced. Many organisms secrete very active xylanase enzymes but the levels of production are low and therefore not suitable for industrial production. Anaerobic fungi are typical examples that produce highly active biomass-degrading enzymes but they are not suitable for production of these enzymes under industrial settings because the amount of enzymes produced is low and cultivation under anaerobic conditions is required. To overcome these challenges, we have developed strategies to genetically engineer the genes of the anaerobic fungal xylanases into the industrially used fungus Trichoderma reesei. The current research demonstrated for the first time the production of anaerobic fungal enzymes by T. reesei. The xylanase over-produced by T. reesei will be used for biomass saccharification and other industrial application tests.
The AT-rich xylanase A gene (xynA) of the anaerobic fungus Orpinomyces sp. strain PC-2 codes for a polypeptide comprising a glycoside hydrolase family 11 catalytic domain linked by a hinge to two docking domains. The catalytic domain-coding region was used for the heterologous production of a xylanase by 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 of the fusion xylanase in culture supernatants by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content could be attributed to the transcript stabilization. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed secretion of the Orpinomyces xylanase catalytic domain. Both the fusion (40 kDa) and the fully processed (28 kDa) forms displayed enzymatic properties of highly active family 11 xylanases. Amino-terminal sequencing analysis of the 28-kDa xylanase indicated that both the R and the Kex2-like KR sites were recognized during the secretion, resulting in a mixture of two amino-termini for the 28-kDa xylanase. Thus, we demonstrated that genes of anaerobic fungi coding for highly active glycoside hydrolases can be efficiently expressed in H. jecorina.