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.

Microbial Conjugation Studies of Licochalcones and Xanthohumol

“…Results obtained from this study showed that A. coerulea is capable of sulfation of chalcones and exhibits parallels between microbial and mammalian metabolism not only in phase I but also in phase II metabolism [21,31]. It is widely known that the phase II sulfation reactions are mediated by the enzyme sulfotransferases, which catalyze the transfer of sulfonate group from the active sulfate to the substrates containing hydroxyl groups [45]. Thus, the sulfate metabolites of licochalcones and xanthohumol were supposed to be produced by the enzyme sulfotransferase from A. coeralea.…”

A Unique Sulfotransferase-Involving Strigolactone Biosynthetic Route in Sorghum

“…Like other plant SOTs, LGS1 is predicted to be localized in cytoplasm. Cytosolic SOTs contain several conserved PAPS-binding motifs, including the one interacts with 5′-phosphate of PAPS (TYPKSGT), 3′-phosphate of PAPS (YxxRNxxDxxVS), and nucleotide of PAPS (GxxGxxK/R) ( Xie et al, 2020 ). Multiple sequence alignment indicates that LGS1 contains these motifs, but with some variations (SLPKSGT and YxxRExxDxxVS, respectively) ( Supplementary Figure 10 ).…”

“…LGS1 contains the highly conserved histidine residues (H216) ( Landi and Esposito, 2020 ) and moderately conserved histidine residues (H317A) ( Supplementary Figure 10 ), which likely act as a base to remove the proton from the substrate hydroxyl group, thereby forming an oxygen anion, and then attacking the sulfo group of PAPS to complete the transfer of the sulfo group. To determine whether these residues play a key role in catalysis, we conducted site-directed mutagenesis on residues likely act as a catalytic base (H216A, H317A) or crucial for PAPS binding (K148A, Y247F) ( Xie et al, 2020 ). While LGS1 H 216 A (resulting strain: YSL8f, Supplementary Table 3 ) exhibited same activity as wild type LGS1, replacing LGS1 with LGS1 K 148 A , LGS1 Y 247 F , and LGS1 H 317 A in ECL/YSL8a (resulting strain: YSL8g-i, Supplementary Table 3 ) completely abolished the synthesis of 4DO and 5DS ( Supplementary Figure 11 ), implying that these residues are critical to the catalytic activity of LGS1 ( Supplementary Figure 11 ).…”

A unique sulfotransferase-involving strigolactone biosynthetic route in Sorghum

“…Similar to other plant SOTs, LGS1 is predicted to be localized in cytoplasm. Cytosolic SOTs contain several conserved PAPS-binding motifs, including the one interacts with 5′-phosphate of PAPS (TYPKSGT), 3’-phosphate of PAPS (YxxRNxxDxxVS), and nucleotide of PAPS (GxxGxxK/R) 26 . Multiple sequence alignment indicates that LGS1 contains these motifs, but with some variations ( SL PKSGT and YxxR E xxDxxVS, respectively) (Figure S10).…”

“…LGS1 contains the highly conserved histidine residues (H216) 27 and moderately conserved histidine residues (H317A) (Figure S10) which likely act as a base to remove the proton from the substrate hydroxyl group, thereby forming an oxygen anion, and then attacking the sulfo group of PAPS to complete the transfer of the sulfo group. To determine whether these residues play a key role in catalysis, we conducted site directed mutagenesis on residues likely act as a catalytic base (H216A, H317A) or crucial for PAPS binding (K148A, Y247F) 26 . While LGS1 H216A (resulting strain: YSL8f , Table S2) exhibited same activity as wildtype LGS1, replacing LGS1 with LGS1 K148A , LGS1 Y247F , and LGS1 H317A in ECL/YSL8a (resulting strain: YSL8g-i , Table S2) completely abolished the synthesis of 4DO and 5DS (Figure S11), implying that these residues are critical to the catalytic activity of LGS1 (Figure S11).…”