Discovery of a novel translation-machinery-associated protein that positively correlates with cellulase production

Abstract

Background

The production of cellulases by filamentous fungi is a crucial aspect of sustainable bioproduction from renewable lignocellulosic biomass. Following the transcription of cellulase genes in the nucleus, a complex pathway involving translation, folding, and secretion is required to produce extracellular cellulases. Most studies about cellulase production have focused on examining transcriptional regulatory mechanisms and enhancement of enzyme gene levels; comparatively, little is known about protein translation and secretion for cellulase production.

Results

A translation-machinery-associated (TMA) protein PoTma15 was identified in cellulosic Penicillium oxalicum. The PoTma15 is conserved in various filamentous fungi, but not in yeast, plants, or animals. All homologous proteins of PoTma15 have previously been uncharacterized. PoTma15 was initially thought to be one of the putative interactors of transcription factor PoXlnR, as it was preyed by tandem affinity purification (TAP) coupled with the mass spectrometry (TAP–MS) technique using PoXlnR as the bait. Subsequent research revealed that PoTma15 is associated with the translation machinery. The top three proteins associated with PoTma15 are orthologs of Saccharomyces cerevisiae translation-machinery-associated protein (Tma19), translation elongation factor eIF5A, and ribosomal protein S28, respectively. PoTma15 is widely distributed in fungal hyphae and positively correlates with the production of cellulases and extracellular proteins. Deleting the Potma15 gene (Δtma15) decreased cellulase production, while overexpressing the Potma15 gene (OEtma15) increased cellulase production. However, the Δtma15 mutant was not observed to have downregulated transcript levels of major (hemi)cellulase and amylase genes, compared to the P. oxalicum wild type (WT). The production of extracellular cellulases and extracellular proteins of the Δtma15 mutant was less affected by cycloheximide, an inhibitor of eukaryotic translation elongation, compared to the WT strain and OEtma15 mutant, suggesting a stronger resistance to the translation-inhibiting effects of cycloheximide in the Δtma15 mutant. The results demonstrate that PoTma15 is a translation-machinery-associated protein that affects translation elongation and, consequently, the production of enzyme proteins.

Conclusions

PoTma15 is the first TMA protein characterized in cellulosic filamentous fungi and the first TMA protein used in fungi to increase cellulase production. PoTma15’s role in the production of cellulases and total extracellular proteins suggests that not only can it be used to widen the cellulase production pathway, but can even be engineered as a target to improve the production of other heterologous protein or bioproducts using filamentous fungi as cell factories in the future.