Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

Abstract

Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley-Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

Keywords: Myceliophthora; galactose transport; galactose utilization; metabolic engineering; transcriptomic profiles.

FIGURE 1

Transcriptional profiling of the genes involved in galactose breakdown in M. thermophila. Firebrick, highest expression; green, lowest expression. GalK, galactokinase; Gal7, galactose-1-phosphate uridyl transferase; Gal10, UDP-glucose-4-epimerase; Pgm2, phosphoglucomutase; Xr, xylose reductase; Ldh, L-arabinitol 4-dehydrogenase; Xhr, L-xylo-3-hexulose reductase; Xdh, xylitol dehydrogenase; Hxk1, Hexokinase-1. Detailed data were shown in Supplementary Table 3.

FIGURE 2

Physiological characterization of M. thermophila strains. (A) Assay of consumption rate of galactose. (B) Dry cell weight of culture of strain OEgal2 grown on galactose for 6 days. (C) Time course of galactose utilization in shaking flasks. (D) Sensitivities of the strain OEgal2ΔgalK to oxidative stress, high osmotic stress, and cell wall disturbance. Mature spore was inoculated onto agar plates alone or supplemented with H₂O₂ (1 mM), NaCl (0.5 M), or Calcofluor White (20 μg/ml) and incubated at 35°C for 6 days. Values and error bars represent means and standard deviations of independent triplicate experiments, respectively.

FIGURE 3

Transcriptional analysis of the strains DeltagalK and OEgal2DeltagalK when responding to galactose. (A) Expression levels of the genes involved in the oxide-reductive pathway. (B) Venn diagram of comparison of transcriptomes of M. thermophila strains grown on galactose. (C) Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the 544 genes with upregulated expression levels in strain OEgal2DeltagalK. Detailed data were shown in Supplementary Table 7.

FIGURE 4

Physiological characterizations of M. thermophila strains HW2212 and HW2302 when grown on galactose. (A) Time profile of galactose utilization in shaking flasks. (B) Growth phenotype of the mutants on galactose for 6 days. (C) Assay of the accumulation of intracellular galactonate. Values and error bars represent means and standard deviations of independent triplicate experiments, respectively.

FIGURE 5

Physiological assays of strain HW2506 on galactose. (A) Accumulation of intracellular 2-keto-3deoxygalactonate. (B) Growth profiling of strain HW2506 on agar plate supplemented with 2% galactose after 6 d of incubation. Values and error bars represent means and standard deviations of independent triplicate experiments, respectively.

FIGURE 6

Growth phenotypes of strain HW2705 on galactose. (A) Time course of galactose consumption. (B) Dry cell weight of culture of strain HW2705 grown on galactose for 4 days. Pre-cultured mycelia on glucose were washed with sterile water and transferred into VMM containing 2% galactose for assays of sugar consumption and biomass formation in Erlenmeyer flask. Values and error bars represent means and standard deviations of independent triplicate experiments, respectively.