Microbial soluble aromatic prenyltransferases for engineered biosynthesis.
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ABSTRACT:
SUBMITTER: Chen HP
PROVIDER: S-EPMC7973389 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The linkage of isoprenoid and aromatic moieties, catalyzed by aromatic prenyltransferases (PTases), leads to an impressive diversity of primary and secondary metabolites, including important pharmaceuticals and toxins. A few years ago, a hydroxynaphthalene PTase, NphB, featuring a novel ten-stranded β-barrel fold was identified in Streptomyces sp. strain CL190. This fold, termed the PT-barrel, is formed of five tandem ααββ structural repeats and remained exclusive to the NphB family until its recent discovery in the DMATS family of indole PTases. Members of these two families exist only in fungi and bacteria, and all of them appear to catalyze the prenylation of aromatic substrates involved in secondary metabolism. Sequence comparisons using PSI-BLAST do not yield matches between these two families, suggesting that they may have converged upon the same fold independently. However, we now provide evidence for a common ancestry for the NphB and DMATS families of PTases. We also identify sequence repeats that coincide with the structural repeats in proteins belonging to these two families. Therefore we propose that the PT-barrel arose by amplification of an ancestral ααββ module. In view of their homology and their similarities in structure and function, we propose to group the NphB and DMATS families together into a single superfamily, the PT-barrel superfamily.
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Project description:Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.
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Project description:Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.
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Project description:Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.
| S-EPMC7414894 | biostudies-literature
Project description:Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. Pseudomonas spp. have broad metabolic potential for degrading aromatic compounds. The objective of this study was to develop a “biological funneling” strategy based on genetic modification to convert complex aromatic compounds into cis,cis-muconate (ccMA) using Pseudomonas putida B6-2 and P. brassicacearum MPDS as biocatalysts. The engineered strains B6-2 (B6-2ΔcatBΔsalC) and MPDS (MPDSΔsalC(pUCP18k-catA)) thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA, attaining molar conversions of 95.3% (ccMA/biphenyl) and 100% (ccMA/naphthalene). Under mixed substrates, B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration. Inserting exogenous clusters like bedDC1C2AB and xylCMAB allowed B6-2 recombinant strains to convert benzene and toluene to ccMA. In mixed substrates, constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene; the highest molar conversion rate of ccMA from mixed substrates was 85.2% when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA) incubation with biphenyl and naphthalene. This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.
| S-EPMC10448021 | biostudies-literature
Project description:The UbiA superfamily of intramembrane prenyltransferases catalyzes a key biosynthetic step in the production of ubiquinones, menaquinones, plastoquinones, hemes, chlorophylls, vitamin E, and structural lipids. These lipophilic compounds serve as electron and proton carriers for cellular respiration and photosynthesis, as antioxidants to reduce cell damage, and as structural components of microbial cell walls and membranes. This article reviews the biological functions and enzymatic activities of representative members of the superfamily, focusing on the remarkable recent research progress revealing that the UbiA superfamily is centrally implicated in several important physiological processes and human diseases. Because prenyltransferases in this superfamily have distinctive substrate preferences, two recent crystal structures are compared to illuminate the general mechanism for substrate recognition.
| S-EPMC4911241 | biostudies-literature
Project description:NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.
| S-EPMC2860626 | biostudies-literature
Project description:Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3' prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo, the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3'-prenylnaringenin (3'-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.
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Project description:Modification of natural products with prenyl groups and the ensuing oxidative transformations are important for introducing structural complexity and biological activities. Penigequinolones (1) are potent insecticidal alkaloids that contain a highly modified 10-carbon prenyl group. Here we reveal an iterative prenylation mechanism for installing the 10-carbon unit using two aromatic prenyltransferases (PenI and PenG) present in the gene cluster of 1 from Penicillium thymicola. The initial Friedel-Crafts alkylation is catalyzed by PenI to yield dimethylallyl quinolone 6. The five-carbon side chain is then dehydrogenated by a flavin-dependent monooxygenase to give aryl diene 9, which serves as the electron-rich substrate for a second alkylation with dimethylallyl diphosphate to yield stryrenyl product 10. The completed, oxidized 10-carbon prenyl group then undergoes further structural morphing to yield yaequinolone C (12), the immediate precursor of 1. Our studies have therefore uncovered an unprecedented prenyl chain extension mechanism in natural product biosynthesis.
| S-EPMC4610815 | biostudies-literature