Combinatorial Biosynthesis of Sulfated Benzenediol Lactones with a Phenolic Sulfotransferase from Fusarium graminearum PH-1

Abstract

Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes. Although fungi are prominent biocatalysts and have been used to sulfate drug-like phenolics, no gene encoding a sulfotransferase enzyme has been functionally characterized from these organisms. Here, we identify a phenolic sulfotransferase, FgSULT1, by genome mining from the plant-pathogenic fungus Fusarium graminearum PH-1. We expressed FgSULT1 in a Saccharomyces cerevisiae chassis to modify a broad range of benzenediol lactones and their nonmacrocyclic congeners, together with an anthraquinone, with the resulting unnatural natural product (uNP) sulfates displaying increased solubility. FgSULT1 shares low similarity with known animal and plant sulfotransferases. Instead, it forms a sulfotransferase family with putative bacterial and fungal enzymes for phase II detoxification of xenobiotics and allelochemicals. Among fungi, putative FgSULT1 homologues are encoded in the genomes of Fusarium spp. and a few other genera in nonsyntenic regions, some of which may be related to catabolic sulfur recycling. Computational structure modeling combined with site-directed mutagenesis revealed that FgSULT1 retains the key catalytic residues and the typical fold of characterized animal and plant sulfotransferases. Our work opens the way for the discovery of hitherto unknown fungal sulfotransferases and provides a synthetic biological and enzymatic platform that can be adapted to produce bioactive sulfates, together with sulfate ester standards and probes for masked mycotoxins, precarcinogenic toxins, and xenobiotics.IMPORTANCE Sulfation is an expedient strategy to increase the solubility, bioavailability, and bioactivity of nutraceuticals and clinically important drugs. However, chemical or biological synthesis of sulfoconjugates is challenging. Genome mining, heterologous expression, homology structural modeling, and site-directed mutagenesis identified FgSULT1 of Fusarium graminearum PH-1 as a cytosolic sulfotransferase with the typical fold and active site architecture of characterized animal and plant sulfotransferases, despite low sequence similarity. FgSULT1 homologues are sparse in fungi but form a distinct clade with bacterial sulfotransferases. This study extends the functionally characterized sulfotransferase superfamily to the kingdom Fungi and demonstrates total biosynthetic and biocatalytic synthetic biological platforms to produce unnatural natural product (uNP) sulfoconjugates. Such uNP sulfates may be utilized for drug discovery in human and veterinary medicine and crop protection. Our synthetic biological methods may also be adapted to generate masked mycotoxin standards for food safety and environmental monitoring applications and to expose precarcinogenic xenobiotics.

Keywords: Fusarium; combinatorial biosynthesis; phenolic sulfotransferase.

FIG 1

FgSULT1 is responsible for the sulfation of lasilarin 1. (A) Product profiles (reversed-phase HPLC-HRESIMS traces recorded as total ion chromatograms) of S. cerevisiae BJ5464-NpgA (66) expressing the indicated PKSs and FgSULT1 from F. graminearum PH-1 (upper two traces) or in vitro biocatalytic transformation of lasilarin 1 by the purified recombinant FgSULT1 enzyme with or without the sulfo group donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) (lower two traces). (B) HRESIMS/MS spectra of lasilarin 1 and its sulfate esters 1a and 1b. (C) Structures of lasilarin 1 and its sulfate esters 1a and 1b.

FIG 2

Structures of BDL and anthraquinone congeners that are sulfated by FgSULT1. The 2,4-dihydroxybenzaldehyde motif shared by these compounds is highlighted in blue. Table S8 in reference lists the PKS pairs whose expression in yeast affords compounds 1 to 13. Fig. S3 in reference shows additional model substrates investigated, while Table S3 and Fig. S4 in reference provide detailed information on the HPLC-HRESIMS/MS identification of the detected products.

FIG 3

Phylogenetic analysis of FgSULT1. Phylogenetic tree of representative SULTs from animals (Homo sapiens, Mus musculus, Rattus norvegicus, and Danio rerio), a plant (Arabidopsis thaliana), bacteria, and fungi reconstructed using the maximum likelihood method. The Fusarium spp. subfamily of predicted SULTs (>65% identity to FgSULT1; >95% coverage) and the Fungi/Bacteria SULT family (>45% identity to FgSULT1; >90% coverage) are indicated by salmon and purple arcs, respectively. Other sulfotransferase domain-containing hypothetical proteins from fungi (23 to 27% identity to FgSULT1, labeled with a blue arc) form a sister clade to animal SULTs. The origins of the enzymes are color-coded as indicated. Numbers on branches show the percentage bootstrap support (when >50%) for each branch point, based on 1,000 pseudoreplicates. The log-likelihood of the phylogenetic tree is −12781.27. The substitution model used the Jones-Taylor-Thornton (JTT) model with uniform rates.

FIG 4

Sequence analysis and homology structure modeling of FgSULT1. (A) Sequence alignment of FgSULT1 with characterized SULTs from the mammalian SULT1 family, the plant SOT5, SOT12, SOT13, and SOT18 enzymes, and the prokaryotic SULT StaL. Black boxes show PAPS binding motifs, the gray box indicates the KTVE dimerization motif in animal SULTs, and additional colored boxes show the loop regions that gate the binding pocket in different SULTs (loops 1a, 1b, 2, and 3 as yellow, blue, purple and green boxes, respectively). Residues in the catalytic center that form hydrogen bonds with PAPS or the substrate are indicated with stars. FgSULT1 amino acid numbering is shown on the right. Amino acids conserved in >70% of the selected proteins are marked in red. (B) Structure superimposition of FgSULT1 (cartoon in blue) with the human cytosolic sulfotransferase SULT1B1 (3CKL, cartoon in sandy brown), including the substrates resveratrol and PAPS (shown as sticks). (C) Close-up of the substrate binding area of FgSULT1, superimposed with resveratrol (cyan sticks) and PAPS (gold sticks) from SULT1B1 (3CKL). Conserved active site residues H101 and K99 are shown as red sticks. Loops 1a, 1b, 2, and 3 are color-coordinated in panels A to C.