Unsymmetrically Regioselective Homodimerization Depends on the Subcellular Colocalization of Laccase/Fasciclin Protein in the Biosynthesis of Phlegmacins

Abstract

Phlegmacins are homodimeric dihydroanthracenone natural products featuring two torosachrysone monomers unsymmetrically conjugated by 7,10′-coupling. Herein, we report the identification and characterization of the biosynthetic gene cluster of phlegmacins in ascomycete Talaromyces sp. F08Z-0631. On the basis of the heterologous reconstitution of the phlegmacin pathway in Aspergillus oryzae, we demonstrated an unprecedented laccase-involved unsymmetrically regioselective oxidative coupling reaction. The association of laccase PhlC and the fasciclin partner protein PhlB was verified to be indispensable for the coupling activity. Intriguingly, both proteins can be transferred and located independently at the mitochondrial membrane. Notably, only their subcellular colocalization led to the occurrence of oxidative dimerization. These observations add new insights into the poorly understood catalytic mechanisms of various laccases involved in the biosynthesis of secondary metabolites, particularly those functioning with variable partners.

Synthetic Biology‐based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities

“…However, it shows no sequence identity (Table S4), implying their different functions to diversify class B PQs. Based on the above data and previous studies, [15,18b,20b] the biosynthetic pathways of all typical class B PQs are proposed (Figure 4A).…”

“…It seems that the addition of CTB11 has no contribution for the formation of ent ‐ 3 in our assay (Figure 3A, x and xi). Recent studies have revealed that fasciclin proteins are membrane proteins and only the subcellular colocalization with laccase could carry out the oxidative dimerization for PQs biosynthesis [20b] . Thus, it indicates that our truncated version of CTB11 loses its regio‐ and stereo‐selective function.…”

“…JNU001 or in vitro reconstitution using purified CTB proteins (Figures 2B and 3A). Since fasciclin proteins control the stereo‐selectivity in phenolic coupling, [20b] and unnatural (+)‐phleichrome ( ent ‐3 ; M helicity), rather than natural phleichrome ( 3 ; P helicity) from C. phlei , [5c] was isolated from the CTB deletion strains (Figures 2B and 4A, i and ii), it indicates that fasciclin proteins CTB11 and ple6 could be swapped to design unnatural PQs with distinct axial chirality (Figure 4A). As for the HYP BGC ( HYP1 ‐ 8 , 11 , 12 and ORF1 , 2 ) and the elc BGC ( elcA ‐ H ) responsible for the biosynthesis of hypocrellin A and elsinochrome A (Figure S19, Table S4), respectively, they also have all the genes necessary for the formation of PQs core (rings A−E).…”

“…According to the recent study, oxidoreductase CTB5 and laccase CTB12 work in tandem to form the core structure of PQ, [18b] a significant step forward in our understanding of PQ biosynthesis. Further research suggests that the fasciclin protein (CTB11) is crucial for laccase's activity (CTB12) in performing the phenol‐coupling reaction, [20a,b] and it might play a role in the regio‐ and stereo‐selective coupling of PQs [15a,18b] . Following the formation of the PQ core, branching pathways help diversify the range of natural PQs.…”

Production of the antifungal biopesticide physcion through the combination of microbial fermentation and chemical post-treatment

“…Similar to that previously described (Zhao et al 2022 ), transformation of A. oryzae NSAR1 was performed by a protoplast–polyethylene glycol method. The spore suspension of A. oryzae NSAR1 was seeded in 10 mL DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO 4 ·7H 2 O, 0.5% KH 2 PO 4 , without pH adjustment), cultured for 2 days at 30 °C and 150 rpm, and then transferred into 100 mL DPY medium for another day.…”