Development of a FungalBraid Penicillium expansum-based expression system for the production of antifungal proteins in fungal biofactories

Abstract

Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.

Fig. 1

Schematic diagrams of FungalBraid transcriptional units (TU) and binary vectors for the expression of afp genes under the control of either the afpA or paf cassettes. A. Binary assembly of TUs FB112 and FB146 with hygromycin (hph) resistant marker (FB003) and geneticin (nptII) resistant marker (FB009) to obtain the final binary vectors to transform P. digitatum (FB114 and FB158) and P. chrysogenum (FB115 and FB159) for the production of PeAfpA. B. Binary assembly of TUs FB230 and FB033 with FB003 and FB009 to obtain the final binary vectors to transform P. digitatum (FB036 and FB244) and P. chrysogenum (FB116 and FB245) for the production of PdAfpB. FB033 is described in Hernanz‐Koers et al. (2018).

Fig. 2

Analyses of P. chrysogenum transformants for PeAfpA production with either the afpA or the paf cassette. A. Schematic diagram of the binary vectors FB115 and FB159 used for P. chrysogenum transformation. B. SDS‐PAGE (top) and western blot analyses (bottom) of pure PeAfpA (2 µg) and growth supernatants of recombinant strains (10 µl of 10× supernatants loaded per lane) obtained by either FB115 (left) or FB159 (right) transformation. SDS‐PAGE analyses were visualized by Coomassie blue staining; M: SeeBlue^® Pre‐stained protein standard. Immunoblot analyses were performed using specific anti‐PeAfpA antibody. Parental strain Δpaf was loaded as a negative control. Positive PeAfpA producing strains (PCMG11522, PCMG11532 and PCMG11552) are highlighted in red. C. Evaluation of afpA gene copy number in the different PeAfpA producing strains by qPCR. The Ct signal of afpA and L18a was normalized to that of β‐tub. Results are presented as mean values ± standard deviation (SD) of three technical replicates. Under this experimental design, the resulting gene copy number is expected to be 1 for afpA in CMP‐1, and ≥ 1 for afpA in P. chrysogenum transformants. D. Colony morphology of P. chrysogenum PeAfpA producing strains PCMG11522, PCMG11532 and PCMG11552 compared to the wild‐type Q176 and the parental strain Δpaf after 6 days of growth on PDA and PcMM plates.

Fig. 3

Analyses of P. digitatum transformants for PeAfpA production using either the afpA or the paf cassette. A. Schematic diagram of the binary vectors FB114 and FB158 used for P. digitatum transformation. B. SDS‐PAGE (top) and western blot analysis (bottom) of pure PeAfpA (2 µg) and growth supernatants of recombinant strains (10 µl of 10× supernatants loaded per lane) obtained by either FB114 or FB158 transformation. SDS‐PAGE analyses were visualized by Coomassie blue staining; M: SeeBlue^® Pre‐stained protein standard. Immunoblot analyses were performed using specific anti‐PeAfpA antibody. Parental strain CECT 20796 was loaded as a negative control. Positive PeAfpA producing strains (PDGL11412, PDGL11432 and PDMG11442) are highlighted in red. C. Evaluation of afpA gene copy number in the different PeAfpA‐producing strains by qPCR. The Ct signal of afpA and L18a was normalized to that of β‐tub. Results are presented as mean values ± SD of three technical replicates. Under this experimental design, the resulting gene copy number is expected to be 1 for afpA in CMP‐1, and ≥ 1 for afpA in P. digitatum transformants. D. Colony morphology of P. digitatum PeAfpA producing strains PDGL11412, PDGL11432 and PDGL11442 compared to the parental strain CECT 20796 after 7 days of growth on PDA and PdMM plates.

Fig. 4

Production and identification of PeAfpA in wild‐type P. expansum CMP‐1 and recombinant P. chrysogenum PCMG11552 strains. A. SDS‐PAGE (top) and western blot analysis (bottom) of 10 µl of 5× supernatants of strains grown in P. chrysogenum minimal medium (PcMM) for 3, 5, 7 and 10 days. One µg of pure PeAfpA was added as control. SDS‐PAGE analysis was visualized by Coomassie blue staining; M: SeeBlue^® Pre‐stained protein standard. Immunoblot analysis was performed using specific anti‐PeAfpA antibody. B. Peptide mass fingerprinting (PMF) of the recombinant PeAfpA protein purified from PCMG11552 grown in PcMM for 5 days. Peptides obtained by PMF covered 49% of PeAfpA primary sequence (top). C. Dose–response curve comparing the antifungal activity of native (red circles) and recombinant PeAfpA (blue circles) against P. digitatum. Plotted data are mean values ± SD of triplicate samples after 48 h at 25°C.

Fig. 5

Analyses of P. chrysogenum transformants for PdAfpB production using either the afpA or the paf cassette. A. Schematic diagram of the binary vectors FB245 and FB116 used for P. chrysogenum transformation. B. SDS‐PAGE (top) and western blot analyses (bottom) of pure PdAfpB (2 µg) and growth supernatants of recombinant strains (10 µl of 10× supernatants loaded per lane) obtained by either FB245 (left) or FB116 (right) transformation. SDS‐PAGE analyses were visualized by Coomassie blue staining; M: SeeBlue^® Pre‐stained protein standard. Immunoblot analyses were performed using specific anti‐PAFB antibody. Parental strain Δpaf was loaded as a negative control. Positive PdAfpB producing strains (PCMG11612 and PCMG11613) are highlighted in green. C. Evaluation of afpB gene copy number in the different PdAfpB producing strains by qPCR. The Ct signal of afpB and L18a was normalized to that of β‐tub. Results are presented as mean values ± SD of three technical replicates. Under this experimental design, the resulting gene copy number is expected to be 1 for afpB in CECT 20796, and ≥ 1 for afpB in P. chrysogenum transformants. D. Colony morphology of P. chrysogenum PdAfpB producing strains PCMG11612 and PCMG11613 compared to the wild‐type Q176 and the parental strain Δpaf after 7 days of growth on PDA and PcMM plates.

Fig. 6

Analyses of P. digitatum transformants for PdAfpB production using the afpA cassette. A. Schematic diagram of the binary vector FB244 used for P. digitatum transformation. B. SDS‐PAGE (top) and western blot analyses (bottom) of pure PdAfpB (2 µg) and growth supernatants of recombinant strains (10 µl of 10× supernatants loaded per lane) obtained after transformation with FB244. SDS‐PAGE analyses were visualized by Coomassie blue staining; M: SeeBlue^® Pre‐stained protein standard. Immunoblot analysis was performed using specific anti‐PAFB antibody. Parental strain CECT 20796 was loaded as a negative control. Positive PdAfpB producing strains (PDAL24425, PDAL24441 and PDAL24444) are highlighted in red. C. Evaluation of afpB gene copy number in the different PdAfpB producing strains by qPCR. The Ct signal of afpB and L18a was normalized to that of β‐tub. Results are presented as mean values ± SD of three technical replicates. Under this experimental design, the resulting gene copy number is expected to be 1 for afpB in CECT 20796, and ≥ 2 for afpB in the transformants. D. Colony morphology of P. digitatum PdAfpB producing strains PDAL24425, PDAL24441 and PDAL24444 compared to the parental strain CECT 20796 after 7 days of growth on PDA and PdMM plates.

Fig. 7

Luciferase assay for testing PafpA and Ppaf strength in Penicillium. A. Schematic diagram of TU assemblies to drive the expression of the luciferase (luc) gene under the control of PafpA or Ppaf (FB258 and FB259) and the Nanoluciferase (Nanoluc) gene under the control of PgpdA (FB312). Final vectors obtained with geneticin (nptII) resistant marker (FB009) were used for transformation of P. chrysogenum and P. digitatum (FB323 and FB324). An insulator sequence (GB3458) was used to allow the binary assembly of the plasmids containing the luc TU with the plasmid containing Nanoluc TU and geneticin resistant marker. B. Luciferase/Nanoluciferase signal ratio of 3 independent transformants for each construct in P. digitatum and P. chrysogenum at 2 days of growth in minimal medium (PdMM or PcMM, respectively). Values are represented as the mean ± standard error (SE). Asterisks (*) denote statistically significant differences in comparison to control values (ANOVA and Tukey’s HSD test, P < 0.05). C. Luciferase/Nanoluciferase signal ratio of a selected transformant for each construct in P. digitatum and P. chrysogenum at 2, 5 and 10 days of growth in PdMM or PcMM, respectively. Values are the means ± SE from three independent replicates. Asterisks (*) denote statistically significant differences between promoters at each time‐point (P < 0.05, Student’s t‐test).