Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina

Abstract

The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function.

FIG. 1.

(Right side) Utilization of carbon sources by H. jecorina QM 6a (solid line) and the two early cellulase mutants QM 9123 (triangles) and QM 9414 (black circles). The order of the carbon sources is the rank of the growth on 95 carbon sources and water, based on the A₇₅₀ value at 48 h for strain QM 6a. Standard deviations are given by error bars. (Left side) Joining cluster analysis applied to carbon sources based on their profiles at 12, 18, 24, 36, 42, and 48 h. Numbers on this side are the same as those on the right side and indicate the compound.

FIG. 2.

Growth curves of carbon sources belonging to clusters I to IV in Fig. 1. A single line corresponds to one carbon source.

FIG. 3.

Growth of H. jecorina QM 6a (•), QM 9123 (□), and QM 9414 (○) on carbon sources, for which statistically significant differences among them were detected. (A) Adonitol; (B) 2-keto-d-gluconic acid; (C) γ-aminobutyric acid; (D) salicin; (E) sorbitol; (F) saccharic acid. Standard deviations are given by vertical bars. Values with different letters are significantly different (ANOVA, post hoc Tukey HSD test, P < 0.05).

FIG. 4.

Utilization of 95 carbon sources by various wild isolates of H. jecorina. The order of carbon sources corresponds to that in Fig. 1 (H. jecorina QM 6a). The strains used were G.J.S 85-236 (⧫), TUB F-1034 (▪), TUB F-430 (▴), TUB F-733 (⋄), CBS 836.91 (□), and TUB F-1066 (▵). Standard deviations are given by bars. The light gray background corresponds to the variability seen in the early cellulase mutants, taken from Fig. 1.

FIG. 5.

Summed map of global carbon utilization profiles of the ex-type QM 6a strain of H. jecorina, cellulase-negative mutant strain QM 9978; uridine auxotrophic (pyr4) mutant TU-6; early cellulase mutant QM 9414; and two triplets of hygromycin B-resistant transformants (the number of integrated copies is given in roman numerals). The map was composed after several two-way joining cluster analyses applied to carbon sources (i) and fungal strains (ii) as two groups of variables. For the pedigrees and relationships of the strains, see Table 1. Due to the low variability in carbon sources from clusters III and IV, only carbon sources from clusters I and II are shown; the respective growth (A₇₅₀ after 48 h) is given by a corresponding color as indicated in the color scale.