Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α‐l‐Arabinofuranosidases in Penicillium oxalicum

Abstract

α‐l‐Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α‐l‐arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l‐arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V, is constructed to improve the production of α‐l‐arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V‐overexpressing strain can synthesize α‐l‐arabinofuranosidase in the absence of an inducer and shows a 54.1‐fold increase in α‐l‐arabinofuranosidase production and a 7.4‐fold increase in α‐galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α‐l‐arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α‐ l‐arabinofuranosidase production.

Gene Regulatory Networks of Penicillium echinulatum 2HH and Penicillium oxalicum 114-2 Inferred by a Computational Biology Approach

“…Zn2Cys6 binuclear clusters have been identified in all fungal species analyzed so far. For example, Zn2Cys6 zinc finger proteins, such as AmyR (PDE_03964), XlnR (PDE_07674) (Li et al, 2015), and AraR (PDE_04461) (Gao et al, 2019) act in the regulation of carbon metabolism in P. oxalicum 114-2. Based on this knowledge, this domain could be considered ubiquitous to fungi.…”

Combinatorial Engineering of Transcriptional Activators in Penicillium oxalicum for Improved Production of Corn-Fiber-Degrading Enzymes

“…As previously reported, expression of the constitutively active forms of transcriptional activators ClrB, XlnR, and AraR could enhance the production of multiple lignocellulolytic enzymes in P. oxalicum. − The engineering of TFs also changed the composition of extracellular proteins to different extents, which might affect their performance in corn fiber saccharification. Here, the regulatory interactions between TFs and lignocellulolytic enzyme genes were studied in detail.…”

“…Then, each strain was compared with the parent for extracellular lignocellulolytic enzyme activities and genome-wide transcriptional changes (Figure A). To exclude the interference of inducible carbon sources in lignocellulolytic enzyme gene expression, the strains were cultivated in the medium without a carbon source, where the constitutively active mutants of TFs could trigger the expression of their target genes in the absence of an inducer. ,, …”

“…The filter paper enzyme (FPase), endoglucanase (CMCase), cellobiohydrolase ( p NPCase), β-glucosidase ( p NPGase), xylanase, β-xylosidase ( p NPXase), α-arabinofuranosidase ( p NPAase), α-galactosidase ( p NPGALase), and amylase activities of culture supernatants were measured using a Whatman No. 1 filter paper, sodium carboxymethyl cellulose, p -nitrophenyl-β- d -cellobioside, p -nitrophenyl-β- d -glucoside, insoluble xylan, p -nitrophenyl-β- d -xylopyranoside, p -nitrophenyl-α- l -arabinofuranoside, p -nitrophenyl-α- d -galactopyranoside, and soluble starch as the substrates, respectively, as described in detail previously. , One enzyme activity unit (U) was defined as the amount of enzyme that liberates 1 μmol of glucose (xylose for xylanase) equivalent or p -nitrophenol per minute under the assay conditions. Protein concentrations were determined using the modified Bradford reagent (Sangon, China) with bovine serum albumin as a standard.…”

“…oxalicum is controlled by a set of transcriptional factors (TFs). − Previously, we have engineered three of the transcriptional activators, namely ClrB, XlnR, and AraR, using point mutation or domain exchange strategies. In the engineered strains, the inducer-independent expression of cellulase and hemicellulase genes was achieved, and the production of enzymes in inducing lignocellulosic media was also enhanced. − However, the performance of the enzymes produced by the wild-type and engineered P. oxalicum strains in corn fiber degradation remains unknown.…”