Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi

Abstract

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.

FIGURE 1.

Localization of A. nidulans BioF protein. A, the bioF open reading frame was fused to GFP, and the chimeric gene was expressed from the constitutive gpdA promoter. The BioF C-terminal amino acids are ARL. Construct GFP-BioF-FL contains full-length bioF fused to GFP (as shown in A), whereas construct GFP-BioF-TR contains bioF lacking the codons for the last three C-terminal amino acids (ARL) fused to GFP. An, A. nidulans. B, vegetative hyphae of stable transformants expressing a DsRed-PTS1 peroxisomal marker and either GFP-BioF-FL (panels a–d) or GFP-BioF-TR (panels e–h) were analyzed by confocal microscopy. Fluorescence was acquired in the green channel (GFP; panels b and f) and red channel (panels c and g). Light transmission (Trans.) images (panels a and e) and the superimposition of green and red fluorescence (panels d and h) are shown. Scale bars = 20 μm.

FIGURE 2.

Complementation of A. nidulans bioF deletion mutant with GFP-bioF chimeric constructs. A. nidulans biotin-prototrophic strain FGSC A1145 (dish 1), the ΔbioF mutant (dish 2), and the ΔbioF mutant transformed with either the GFP-BioF-FL (dish 3) or GFP-BioF-TR (dish 4) construct were grown on minimal medium deficient in biotin and supplemented with avidin (upper) or supplemented with biotin (lower).

FIGURE 3.

Complementation of E. coli biotin-auxotrophic mutant bioF103 with GFP-bioF constructs. The E. coli mutant bioF103 strain was transformed with a full-length A. nidulans bioF cDNA cloned in vector pTRC99A (sector 1), empty vector pTRC99A (sector 2), and either the GFP-BioF-FL (sector 3) or GFP-BioF-TR (sector 4) chimeric construct, both in pTRC99A. The medium in the left dish was biotin-deficient and contained avidin, whereas that in the right dish was supplemented with biotin.

FIGURE 4.

A. nidulans mutants in the peroxisomal β-oxidation cycle require biotin for growth. Spores (10³) from a control (CTL) biotin-prototrophic strain (FGSC A1145) and from biA1, ΔbioF, ΔaoxA, ΔaoxB, ΔpexE, and ΔfoxA mutants were point-inoculated individually in wells of a 24-well microtiter plate containing either minimal medium supplemented with biotin (lower row) or biotin-deficient minimal medium supplemented with avidin (upper row) or with avidin and pimelic acid (middle row) and incubated for 1 day at 37 °C and for 2 days at 28 °C. As reported previously (26), the ΔpexE mutant showed reduced sporulation compared with all other strains, even when grown on medium containing biotin, explaining the paler appearance of the ΔpexE colony. However, the mycelial growth rate of the ΔpexE mutant (colony diameter and mycelial density) is comparable with that of the wild-type control strain on biotin-supplemented medium.

FIGURE 5.

β-Oxidation cycle is not required for biotin synthesis in E. coli. Cells were grown on medium supplemented with biotin (right) or on biotin-depleted medium containing avidin (left). The strains used were biotin-auxotrophic ΔbioD (sector 1); control BW25113 (sector 2); and deletion mutants ΔfadA (sector 3), ΔfadB (sector 4), ΔfadE (sector 5), ΔfadK (sector 6), ΔfadJ (sector 7), and ΔfadI (sector 8). Plates were incubated under aerobic (upper) or anaerobic (lower) conditions.

FIGURE 6.

Peroxisomal β-oxidation cycle or import of peroxisomal proteins is dispensable for biotin synthesis in S. cerevisiae. A, strains transformed with either empty vector p416 or a plasmid carrying the BIO6/BIO1 gene cluster (BIO) were grown on medium containing biotin (right) or on biotin-depleted medium supplemented with avidin (left). The strain/plasmid combinations were BY4742/p416 (sector 1), BY4742/BIO (sector 2), pox1Δ0/p416 (sector 3), pox1Δ0/BIO (sector 4), fox2Δ0/p416 (sector 5), fox2Δ0/BIO (sector 6), pot1Δ0/p416 (sector 7), pot1Δ0/BIO (sector 8), pex5Δ0/p416 (sector 9), pex5Δ0/BIO (sector 10), pex7Δ0/p416 (sector 11), and pex7Δ0/BIO (sector 12). B, growth kinetics of strains BY4742/BIO (WT), pox1Δ0/BIO, and fox2Δ0/BIO in liquid biotin-depleted medium supplemented with avidin. Absorbance at 600 nm (OD 600; 0.6-cm light path) was recorded. Error bars represent S.E. (n = 6).