Physiological Characteristics and Comparative Secretome Analysis of Morchella importuna Grown on Glucose, Rice Straw, Sawdust, Wheat Grain, and MIX Substrates

Abstract

Morels (Morchella sp.) are economically important edible macro-fungi, which can grow on various synthetic or semi-synthetic media. However, the complex nutritional metabolism and requirements of these fungi remain ill-defined. This study, based on the plant biomass commonly used in the artificial cultivation of morels, assessed and compared the growth characteristics and extracellular enzymes of Morchella importuna cultivated on glucose, rice straw, sawdust, wheat grain, and a mixture of equal proportions of the three latter plant substrates (MIX). M. importuna could grow on all five tested media but displayed significant variations in mycelial growth rate, biomass, and sclerotium yield on the different media. The most suitable medium for M. importuna was wheat and wheat-containing medium, followed by glucose, while rice straw and sawdust were the least suitable. A total of 268 secretory proteins were identified by liquid chromatography coupled with tandem mass spectrometry detection. Functional classification and label-free comparative analysis of these proteins revealed that carbohydrate-active enzyme (CAZYme) proteins were the predominant component of the secretome of M. importuna, followed by protease, peptidase, and other proteins. The abundances of CAZYme proteins differed among the tested media, ranging from 64% on glucose to 88% on rice straw. The CAZYme classes of glycoside hydrolases and carbohydrate-binding module were enriched in the five secretomes. Furthermore, the enzyme activities of CMCase, lignase, amylase, xylase, pNPCase, and pNPGase were detected during the continuous culture of M. importuna in MIX medium, and the relative expression of the corresponding genes were detected by quantitative real-time PCR. The combined data of growth potential, secretome, extracellular enzyme activity, and gene expression on different substrates inferred that M. importuna was weak in lignocellulose degradation but a good starch decomposer. Specifically, in terms of the degradation of cellulose, the ability to degrade cellulose into oligosaccharides was weaker compared with further degradation into monosaccharides, and this might be the speed-limiting step of cellulose utilization in M. importuna. In addition, M. importuna had a strong ability to decompose various hemicellulose glycosidic bonds, especially α- and β-galactosidase. Only a very few lignin-degradation-related proteins were detected, and these were in low abundance, consistent with the presence of weak lignin degradation ability. Furthermore, the presence of lipase and chitinase implied that M. importuna was capable of decomposition of its own mycelia in vitro. The study provides key data that facilitates a further understanding of the complex nutritional metabolism of M. importuna.

Keywords: CAZYme; amylase; enzyme activity; nutritional metabolism; secretory proteome; substrate utilization.

FIGURE 1

Growth potential of Morchella importuna on different media. Mycelium growth rate (A) and biomass (B) were also measured on the five different media; significant differences are marked by asterisks, *p < 0.05, **p < 0.01. The colony morphology of M. importuna was examined on CYM medium (termed G) (C), glucose-free CYM medium containing 2% rice straw powder (RS) (D), 2% sawdust powder (SD) (E) or 2% wheat grain powder (WG) (F), and media containing equal proportions of rice straw, sawdust, and wheat grain powders (termed MIX) (G).

FIGURE 2

Functional classification of the secretome of Morchella importuna in five different culture media. G, CYM medium; RS, glucose-free CYM containing 2% rice straw powder; SD, glucose-free CYM containing 2% sawdust powder; WG, glucose-free CYM containing 2% wheat grain powder; MIX, media containing equal proportions of rice straw, sawdust, and wheat grain powders.

FIGURE 3

Proportion of the secretome of Morchella importuna in five different culture media. G, CYM medium; RS, glucose-free CYM containing 2% rice straw powder; SD, glucose-free CYM containing 2% sawdust powder; WG, glucose-free CYM containing 2% wheat grain powder; MIX, media containing equal proportions of rice straw, sawdust, and wheat grain powders.

FIGURE 4

Enzyme activities of Morchella importuna on MIX medium.