Tailoring Enzyme-Rich Environmental DNA Clones: A Source of Enzymes for Generating Libraries of Unnatural Natural Products

Abstract

A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) mega-libraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes. Enzymes found on tailoring enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

“…study for isolation of new glycopeptide biosynthetic gene clusters from eDNA libraries identified another sulfotransferase from clone AZ205 with the same regiospecificity as Teg13 (16). Interestingly, the tailoring enzymes found in eDNA-derived gene clusters were used to produce a total of 15 unique sulfated GPA derivatives in both in vitro and in vivo studies (16). The sulfotransferase enzymes within the Teg cluster share ∼60% sequence identity with StaL, and all use the 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor as the source of the sulfate group, converting it to the product 3′-phosphoadenosine 5′-phosphate (PAP) after transfer to the GPA substrate.…”

“…wrote the paper. study for isolation of new glycopeptide biosynthetic gene clusters from eDNA libraries identified another sulfotransferase from clone AZ205 with the same regiospecificity as Teg13 (16). Interestingly, the tailoring enzymes found in eDNA-derived gene clusters were used to produce a total of 15 unique sulfated GPA derivatives in both in vitro and in vivo studies (16).…”

Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay

“…HsS-COMT methyltransferase showed activity with the flavonoids myrcetin [13] and luteolin [15], both similar to the known flavonol substrate quercetin [14]. AtOMT1 methyltransferase showed activity with protocatechuic acid [2] and 4-nitrocatechol [5], which mimic the catechol region of quercetin [14] acted upon by AtOMT1. Last, both HsS-COMT and AtOMT1 showed activity with crocin [26], a carotenoid, with no similarities to previous reported substrates.…”

“…4 Such a gap between empirical data and the number of deposited gene sequences can only increase with mining of metagenomic sequences. 5,6 The need for empirically determined function is especially important for the generation of novel biosynthetic pathways in metabolic engineering. One factor hindering the highthroughput characterization of enzymes, or related pursuits in enzyme engineering, is the available suite of biochemical monitoring methods.…”

Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products

“…S1 and S2) most closely related to the friulimicin (24) biosynthetic gene cluster but with differences in both the megasynthetase biosynthetic machinery and the tailoring enzyme content. The two remaining antibiotic gene clusters are predicted to encode molecules structurally related to the glycopeptides teicoplanin and A47934 (8,25). In each case, the combination of tailoring enzymes seen in these clusters is predicted to result in the production of a previously unknown glycopeptide derivative (Figs.…”