Domain Truncation in Hispidin Synthase Orthologs from Non-Bioluminescent Fungi Does Not Lead to Hispidin Biosynthesis

Abstract

Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence.

Transcriptome Analysis Reveals the Putative Polyketide Synthase Gene Involved in Hispidin Biosynthesis in Sanghuangporus sanghuang

“…The domain architecture of ShPKS1 was AMP-ACP-KS-AT-DH-KR-ACP-ACP. It is similar to the hispidin synthase of H. sublateritium (AMP-ACP-KS-AT-DH-KR-ACP) [ 11 ]. Thus, ShPKS1 is a putative hispidin synthase in S. sanghuang .…”

“…The hispidin synthase (HispS) of bioluminescent fungi has a AMP-ACP-KS-AT-ACP domain architecture. Moreover, the HispS in non-bioluminescent fungi has more DH and KR domains, and its domain architecture is AMP-ACP-KS-AT-DH-KR-ACP [ 11 ]. In the present study, we found that ShPKS1 (AMP-ACP-KS-AT-DH-KR-ACP-ACP) is a putative HispS.…”

“…Hispidin is a type of styrylpyrone, and it has antioxidant [ 1 ], antitumor [ 2 , 3 ], anti-inflammatory [ 4 , 5 ], antiviral [ 6 ], antiallergic [ 7 ], neuroprotective [ 8 , 9 ], antiangiogenic, and hypolipidemic properties [ 10 , 11 ]. Hispidin is a yellow pigment and was first isolated from Inonotus hispidus fungi [ 12 ].…”

“…Subsequently, hispidin has been identified in various fungal species of both the Hymenochaetaceae family, including species in the genera Inonotus ( I. obliquus , I. radiatus , I. xeranticus ), Hymenochaete , Phellinus [ 13 ], and Sanghuangporus [ 14 ], and the Hymenogastraceae family, including the genus Gymnopilus [ 15 ]. Hispidin and hispidin derivatives are also found in other fungal families such as the families Phaeolaceae [ 16 ], Omphalotaceae ( Neonothopanus nambi ) [ 17 ], Strophariaceae ( Hypholoma sublateritium ), and Cortinariaceae ( Cortinarius glaucopus ) [ 11 ]. Furthermore, hispidin derivatives were also identified in some plants, including Equisetum arvense [ 18 ], Pistacia atlantica [ 19 ], Leishmania amazonensis [ 20 ], and Goniothalamus umbrosus [ 21 ].…”

“…Fungal type I PKSs are also classified into aromatic or non-reducing PKSs, partially reducing PKSs, and highly reducing PKSs according to their domain architecture [ 24 ]. Several partially reducing PKSs were confirmed to be related to hispidin biosynthesis in bioluminescent and non-bioluminescent Agaricales [ 11 ]. In plants, a chalcone synthase (CHS) corresponding to a type III PKS has also been reported to be related to styrylpyrone biosynthesis in Piper nigrum [ 25 ].…”