Disruption of zinc finger DNA binding domain in catabolite repressor Mig1 increases growth rate, hyphal branching, and cellulase expression in hypercellulolytic fungus Penicillium funiculosum NCIM1228

Abstract

Background: There is an urgent requirement for second-generation bio-based industries for economical yet efficient enzymatic cocktail to convert diverse cellulosic biomass into fermentable sugars. In our previous study, secretome of Penicillium funiculosum NCIM1228 showed high commercial potential by exhibiting high biomass hydrolyzing efficiency. To develop NCIM1228 further as an industrial workhorse, one of the major genetic interventions needed is global deregulation of cellulolytic genes to achieve higher enzyme production. Mig1 orthologs found in all yeast and filamentous fungi are transcriptional regulators that maintain carbon homeostasis by negatively regulating genes of secondary carbon source utilization. Their disruption has long been known to be beneficial for increasing the production of secreted enzymes for alternate carbon source utilization.

Results: Upon detailed genotypic and phenotypic analysis, we observed that NCIM1228 harbors a truncated yet functional allele of homolog of a well-known catabolite repressor, Mig1. Alleviation of carbon repression in NCIM1228 was attained by replacing functional Mig1¹³⁴ allele with null allele Mig1⁸⁸. P. funiculosum having Mig1⁸⁸ null allele showed better growth characteristics and 1.75-fold better glucose utilization than parent strain. We also showed that visibly small colony size, one of the major characteristics of CCR disruptant strains in filamentous fungi, was not due to retarded growth, but altered hyphal morphology. CCR-disrupted strain PfMig1⁸⁸ showed profuse branching pattern in terminal hyphae resulting in small and compact colonies with compromised filamentous proliferation. We further observed that basal level expression of two major classes of cellulases, namely, cellobiohydrolase and endoglucanase, was regulated by Mig1¹³⁴ in NCIM1228, whereas other two major classes, namely, xylanases and β-glucosidase, were only marginally regulated. Finally, CCR disruption in P. funiculosum NCIM1228 led to prolonged cellulase induction in production medium resulting in twofold increased cellulase activity than the parent strain with maximum secreted protein titer being > 14 g/l.

Conclusions: CCR-disrupted P. funiculosum showed better growth, enhanced carbon source utilization, profuse branching pattern in terminal hyphae, and higher cellulase activity than parent strain. Our findings are particularly important in shedding light on important functions performed by Mig1 in addition to its role as negative regulator of alternate carbon source utilization in filamentous fungi.

Keywords: Carbon catabolite repression; Cellulases; De-repression; Mig1 orthologs; Zinc finger domains.

Fig. 1

Phylogenetic tree of Mig1 orthologs in fungi. Mig1 nucleotide sequences of 41 industrially important fungal species representing 31 orders of fungal kingdom were taken to construct the phylogenetic tree. Mig1 sequence of Saccharomyces cerevisiae was used for rooting the Phylogenetic tree. PfMig1 shares one of the most recently evolved clades along with Penicillium marneffei and Talaromyces cellulolyticus

Fig. 2

PfMig1 has a stop codon at 134th amino acid position. a Diagrammatic representation of putative domains detected in PfMig1. Red line states the presence of nonsense mutation at 134th amino acid position just before the alanine-rich region, thus naming the allele as Mig1¹³⁴. b Sequence alignment of Mig1 protein of closely related fungi with PfMig1. The alignment indicates that the stop codon at 134th position was not due to natural selection, but was rather due to the result of laboratory induced mutation. Sequence from amino acid position 79–139 was taken as query. Red box indicates the 134th position corresponding to stop codon of PfMig1

Fig. 3

Mig1¹³⁴ is a functional allele. a Conidiospores of NCIM1228 carrying Mig1¹³⁴ allele was spotted in duplicate on SC media having 2% Avicel in the absence and presence of 0.5% 2-deoxyglucose (2-DG) and was found sensitive to it. b Conidiospores of NCIM1228 were spotted in duplicate on SC media having 2% glucose in the absence and presence of 1 mM allyl alcohol (AA) and was found resistant to it

Fig. 4

CCR is disrupted in PfMig1⁸⁸. Graphical representation of a Mig1⁸⁸ cassette constructed for homologous recombination and cloned in pCambia1302, and b Truncated Mig1⁸⁸ protein. c PCR amplification products using Mig1 flanking primers. Lane 1 represents DNA ladder, Lane 2 shows amplification of 2248-bp fragment indicating native Mig1 gene and flanking sequence of NCIM1228, and Lane 3 shows amplification of 3035-bp fragment using P1 and P2 primers indicating homologous recombination of PfMig1⁸⁸ cassette leading to loss of native Mig1 sequence. d RACE experiments to confirm the replacement of native gene with Mig1⁸⁸ at RNA level. Lane 1 represents DNA ladder, Lane 2 shows amplification of 1248-bp band representing full-length Mig1 RNA in NCIM1228 and no amplification in Lane 3 shows its absence in PfMig1⁸⁸ transformant, Lanes 4 and 5 represent 5′ RACE where 253-bp fragment from 5′ end of Mig1 RNA was amplified in both NCIM1228 and PfMig1⁸⁸, and Lanes 6 and 7 represent 3′ RACE where NCIM1228 shows amplification of 319-bp fragment representing intact 3′-end of Mig1 RNA, whereas failed 3′ RACE in PfMig1⁸⁸ shows truncated Mig1⁸⁸ RNA. 1-kb Plus DNA ladder (Fermentas) was used as DNA marker. Conidiospores of NCIM1228 and PfMig1⁸⁸ were spotted on e SC media having 2% Avicel in the absence and presence of 0.5% 2-DG, where PfMig1⁸⁸ was found resistant to 2-DG, and on f SC media having 2% glucose in the absence and presence of 1 mM AA, where PfMig1⁸⁸ was found sensitive to 1 mM AA

Fig. 5

PfMig1⁸⁸ features physiognomies different from NCIM1228. a 10 µl of 10⁶ conidiospore suspension of NCIM1228 and PfMig1⁸⁸ were spotted on SC media having different carbon sources and growth of the cells was observed after 48 h. Appearance of colonies on b SC-glucose and c SC-cellobiose plate after 7 days and 14 days. d Mycelial biomass and e secretory protein concentration of PfMig1⁸⁸ relative to NCIM1228 on SC-glucose and SC-cellobiose liquid media after 24 h of cultivation. Total glucose (f) and cellobiose (g) uptake rate when NCIM1228 and PfMig1⁸⁸ were grown in SC liquid media having 1% glucose and 1% cellobiose, respectively. The uptake rate was monitored by measuring residual glucose and cellobiose remaining in the medium at various time intervals starting with equal spore count

Fig. 6

Cellobiose gets hydrolyzed to glucose before utilization. Residual cellobiose and glucose was monitored with respect to time for a NCIM1228 and b PfMig1⁸⁸ strains grown in SC medium having 1% cellobiose

Fig. 7

Presence of PfMig1⁸⁸ allele induces profuse hyphal branching. a Colony morphology of NCIM1228 and PfMig1⁸⁸ after 10 days of incubation. Black arrow denotes the radius of the whole colony, blue arrow denotes the extent of core and middle layer, and red arrow shows highly extended invading layer in NCIM1228 and highly reduced outer layer in PfMig1⁸⁸. The outer layer of NCIM1228 and PfMig1⁸⁸ cells were observed under fluorescence microscope at ×4 magnification (b) and at ×20 magnification (c). In NCIM1228, parallel grouped hyphal structures are shown running throughout the outer layer, whereas no such structures were observed in PfMig1⁸⁸. d Graphical representation of colony morphology observed in NCIM1228 and PfMig1⁸⁸. NCIM1228 colonies are organized around parallel hyphae running from core of the colony toward periphery, whereas no such impeded organization was observed in PfMig1⁸⁸. e Branching pattern observed in terminal hyphae of NCIM1228 and PfMig1⁸⁸ colonies

Fig. 8

Asexual life cycle is not affected in the presence of Mig1⁸⁸ allele. a Microscopic evaluation of (b) conidiophores at ×60 magnification and b germinating spores at ×20 magnification in NCIM1228 and PfMig1⁸⁸. c Percentage germination of spores for NCIM1228 and PfMig1⁸⁸ after 18 h of growth in PD broth. d Percentage viability of NCIM1228 and PfMig1⁸⁸ calculated by plating 50 spores of each strain on PD plates and counting the colonies appeared after 72 h

Fig. 9

Secretome profiling of NCIM1228 and PfMig1⁸⁸ in the presence of glucose. a Upper panel shows SDS-PAGE profile of 10 times diluted secretome of NCIM1228 and PfMig1⁸⁸ grown in Mandel media having varying ratios of glucose and Avicel. Lower panel shows detection of CBHI under different conditions by Western blotting using rabbit anti-CBHI primary antibody and HRPO-conjugated anti-rabbit secondary antibody. Activities for b Avicelase, c CMCase, d xylanase, e β-glucosidase, and f FPase were measured in the secretome of NCIM1228 and PfMig1⁸⁸ when grown under different ratios of glucose and Avicel for 5 days

Fig. 10

Assessing inhibiting concentration of glucose in the presence of inducer for NCIM1228 and PfMig1⁸⁸. Activities for a avicelase, b CMCase, c xylanase, and d β-glucosidase were measured in the secretome of NCIM1228 and PfMig1⁸⁸ when grown under 2% Avicel and glucose concentration ranging from 0 to 2.5%

Fig. 11

Cellulase expression gets de-repressed in PfMig1⁸⁸. a The transcriptional expression of various families of cellobiohydrolase, endoglucanase, β-glucosidase, and xylanase in PfMig1⁸⁸ were measured by quantitative real-time PCR after growing for 24 h in the presence of 4% glucose. The expression levels were normalized to NCIM1228 and plotted. b Transcript levels of various families of cellobiohydrolase, endoglucanase, β-glucosidase, and xylanase in NCIM1228 and PfMig1⁸⁸ in the presence of 4% Avicel. Expression levels are shown in log scale and were normalized to NCIM1228 and PfMig1⁸⁸ grown in the presence of 4% glucose

Fig. 12

Enhanced cellulase secretion and activity was detected in PfMig1⁸⁸ in production medium. a Growth of the fungus in production media was monitored for 7 days by estimating the mycelial proteins by BCA method. RCM in X-axis denotes the amount of protein present in the pellet of medium before inoculation. b Proteins present in the secretome of NCIM1228 (upper panel) and PfMig1⁸⁸ (lower panel) were collected for each day and separated on 10% SDS-PAGE gel followed by staining with Coomassie blue. c Secretome proteins of NCIM1228 and PfMig1⁸⁸ collected for each day was measured by BCA method after exchanging growth medium with citrate–phosphate buffer (pH 4.0). RCM in X-axis denotes the amount of protein present in the supernatant of medium before inoculation. d Total cellulase activity profile represented by FPU/ml of NCIM1228 and PfMig1⁸⁸ during 7 days of cultivation