Transcription Factor NsdD Regulates the Expression of Genes Involved in Plant Biomass-Degrading Enzymes, Conidiation, and Pigment Biosynthesis in Penicillium oxalicum

Abstract

Soil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor, Penicillium oxalicum NsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes in P. oxalicum PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development in Aspergillus In the present study, the regulatory roles of PoxNsdD in P. oxalicum were further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdD strain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70 parental strain. During cultivation, ΔPoxNsdD cultures changed color, from pale orange to brick red, while the ΔPoxKu70 cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed that PoxNsdD dynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover, in vitro binding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering of Poxalicum for potential industrial and medical applications.IMPORTANCE Most filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungus Penicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential of Poxalicum via genetic engineering.

Keywords: Penicillium oxalicum; PoxNsdD; conidiation; pigment biosynthesis; starch-degrading enzyme; transcription factor.

FIG 1

Transcriptomic profiling of the ΔPoxNsdD mutant and the ΔPoxKu70 parental strain during growth in the presence of Avicel as the carbon source. (A) Eukaryotic orthologous group (KOG) annotation of 716 genes in the PoxNsdD regulon (a |log₂ fold change| of ≥1 and a probability of ≥0.8 were used as thresholds). (B) Regulation of 95 genes encoding carbohydrate-active enzymes by PoxNsdD. Red lines represent negative regulation, and green lines with arrowheads represent positive regulation. Blue circles show the degree of regulation. (C) Regulation of 17 genes putatively involved in asexual development (conidiation) in P. oxalicum by PoxNsdD.

FIG 2

Putative biosynthetic gene clusters of known secondary metabolites for which core biosynthetic genes are regulated by PoxNsdD. (A) Cluster 11 = emericellin; (B) cluster 29 = leucinostatins; (C) cluster 33 = roquefortine C/meleagrin; (D) cluster 49 = beauvericin; (E) cluster 51 = cytochalasin; (F) cluster 72 = malbrancheamide; (G) cluster 88 = viridicatumtoxin. Gene IDs labeled in red represent core biosynthetic genes.

FIG 3

Activities of crude enzymes from the PoxNsdD deletion mutant of P. oxalicum following a shift in growth from glucose to soluble starch. Crude enzymes were produced by P. oxalicum strains grown in 1% soluble corn starch as the sole carbon source after a shift from glucose. Enzymatic activity was measured 2 to 4 days after the shift. (A) Raw cassava starch-degrading enzyme (RCSDE) activities. (B) Soluble starch-degrading enzyme (SSDE) activities. Asterisks indicate significant differences (**, P < 0.01; *, P < 0.05) between the ΔPoxNsdD mutant and the ΔPoxKu70 parental strain or the complementary strain (CPoxNsdD), as assessed by Student's t test.

FIG 4

Phenotypic comparison of the ΔPoxNsdD mutant, the complementary strain (CPoxNsdD), and the ΔPoxKu70 parental strain on solid medium plates. (A) Colonies on plates containing PDA, glucose, Avicel, or starch were inoculated at 28°C for 4 days. (B) Schematic diagram of the P. oxalicum strains in panel A. (C) Quantitative analysis of conidiation on solid medium plates containing different carbon sources. P. oxalicum strains were grown on PDA plates at 28°C for 3 days, on glucose plates for 15 days, and on Avicel and starch plates for 10 days. Asterisks indicate significant differences (**, P < 0.01; *, P < 0.05; Student's t test) between the ΔPoxNsdD mutant and the ΔPoxKu70 parental strain or the complementary strain (CPoxNsdD).

FIG 5

Microscopy of hyphae in liquid media containing glucose, Avicel, or starch as the carbon source. Conidiophores are marked by red arrowheads. Bars = 20 μm.

FIG 6

Pigment biosynthesis by the ΔPoxNsdD mutant, the complementary strain (CPoxNsdD), and the ΔPoxKu70 parental strain grown on liquid media. P. oxalicum strains were inoculated directly into liquid modified minimal medium containing glucose, Avicel, or soluble corn starch as the sole carbon source and then cultured at 28°C for 1 to 6 days.

FIG 7

Kinetics of regulation of gene expression by PoxNsdD in P. oxalicum as revealed by real-time quantitative reverse transcription-PCR. (A) Gene expression under induction by soluble corn starch as the sole carbon source at four different time points (4, 12, 24, and 48 h) after shifting of the carbon source. Expression levels were normalized against those of the ΔPoxKu70 parental strain. Asterisks indicate significant differences (**, P < 0.01, *, P < 0.05) between the tested samples and those of the ΔPoxKu70 parental strain, as assessed by Student's t test. (B) Expression of the POX06534/brlA gene in the presence of Avicel. Expression levels were normalized against those of the ΔPoxKu70 parental strain. Asterisks indicate significant differences (**, P < 0.01) between the tested samples and those of the ΔPoxKu70 parental strain, as assessed by Student's t test. (C) Gene expression during vegetative growth and induction of asexual development. For vegetative growth, gene expression was measured at three different time points (12, 24, and 36 h) after the inoculation of spores into medium containing Avicel as the sole carbon source. For asexual development, hyphae grown for 36 h were transferred onto solid medium plates containing Avicel, and gene expression was measured at three different time points (12, 24, and 36 h) after the shift. Expression levels were normalized against those for spores of the ΔPoxNsdD mutant and the ΔPoxKu70 parental strain.

FIG 8

In vitro analysis of binding between the DNA-binding domain of PoxNsdD and the promoter sequences of target genes by electrophoretic mobility shift assay. Each reaction mixture contained 0 to 2.0 μg of Trx-His-S-tagged PoxNsdD_335–494 and about 40 ng of each tested probe. As negative controls, the same amount of purified Trx-His-S fusion protein and BSA were used. POX05989, encoding β-tubulin, was used as a control probe.