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  • The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa.

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa.

Biotechnology for biofuels (2017-06-16)
Nils Thieme, Vincent W Wu, Axel Dietschmann, Asaf A Salamov, Mei Wang, Jenifer Johnson, Vasanth R Singan, Igor V Grigoriev, N Louise Glass, Chris R Somerville, J Philipp Benz
ABSTRACT

Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction. The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of l-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif. PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.

MATERIALS
Product Number
Brand
Product Description

Supelco
Avicel® PH-101, ~50 μm particle size
Sigma-Aldrich
L-(+)-Arabinose, BioUltra, ≥99.5% (sum of enantiomers, HPLC)
Sigma-Aldrich
Pectin from citrus peel, Galacturonic acid ≥74.0 % (dried basis)
Sigma-Aldrich
Polygalacturonic acid, ≥90% (enzymatic)
Sigma-Aldrich
D-(+)-Xylose, ≥99% (GC), BioXtra
Sigma-Aldrich
D-(+)-Galacturonic acid monohydrate, ≥97% (titration)
Vivaspin 6 centrifugal concentrator, MWCO 10 kDa
Sigma-Aldrich
Viscozyme® L, cellulolytic enzyme mixture
Sigma-Aldrich
L-Rhamnose, natural sourced, 99%, FG