In vitro and in vivo characterization of genes involved in mannan degradation in Neurospora crassa

Abstract To better understand the roles of genes involved in mannan degradation in filamentous fungi, in this study we searched, identified, and characterized one putative GH5 endo-β-mannanase (GH5-7) and two putative GH2 mannan-degrading enzymes (GH2-1 and GH2-4) in Neurospora crassa. Real-time RT-PCR analyses showed that the expression levels of these genes were significantly up-regulated when the cells were grown in mannan-containing media where the induction level of gh5-7 was the highest. All three proteins were heterologously expressed and purified. GH5-7 displayed a substrate preference toward galactomannan by showing 10-times higher catalytic efficiency than to linear β-mannan. In contrast, GH2-1 preferred short manno-oligosaccharides or β-mannan as substrates. Compared to the wild type strain, the growth of Δgh5-7 and Δgh5-7Δgh2-4 mutants, but not Δgh2-1, Δgh2-4, and Δgh2-1Δgh2-4 mutants, was poor in the cultures containing glucomannan or galactomannan as the sole carbon source, suggesting that GH5-7 plays a critical role in the utilization of heteromannans in vivo. On the other hand, all the mutants showed significantly slow growth when grown in the medium containing linear β-mannan. Collectively, these results indicate that N. crassa can utilize glucomannan and galactomannan without GH2-1 and GH2-4, but efficient degradation of β-mannan requires a concerted action of three enzymes, GH5-7, GH2-1, and GH2-4.