Steven G. Van Lanen

Microbial genome mining for accelerated natural products discovery: is a renaissance in the making?

Abstract Microbial genome mining is a rapidly developing approach to discover new and novel secondary metabolites for drug discovery. Many advances have been made in the past decade to facilitate genome mining, and these are reviewed in this Special Issue of the Journal of Industrial Microbiology and Biotechnology. In this Introductory Review, we discuss the concept of genome mining and why it is important for the revitalization of natural product discovery; what microbes show the most promise for focused genome mining; how microbial genomes can be mined; how genome mining can be leveraged with other technologies; how progress on genome mining can be accelerated; and who should fund future progress in this promising field. We direct interested readers to more focused reviews on the individual topics in this Special Issue for more detailed summaries on the current state-of-the-art.

A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis

Nonribosomal peptide synthetases (NRPSs) catalyze the biosynthesis of many biologically active peptides and typically are modular, with each extension module minimally consisting of a condensation, an adenylation, and a peptidyl carrier protein domain responsible for incorporation of an amino acid into the growing peptide chain. C-1027 is a chromoprotein antitumor antibiotic whose enediyne chromophore consists of an enediyne core, a deoxy aminosugar, a benzoxazolinate, and a β-amino acid moiety. Bioinformatics analysis suggested that the activation and incorporation of the β-amino acid moiety into C-1027 follows an NRPS mechanism whereby biosynthetic intermediates are tethered to the peptidyl carrier protein SgcC2. Here, we report the biochemical characterization of SgcC5, an NRPS condensation enzyme that catalyzes ester bond formation between the SgcC2-tethered (S)-3-chloro-5-hydroxy-β-tyrosine and (R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core. SgcC5 uses (S)-3-chloro-5-hydroxy-β-tyrosyl-SgcC2 as the donor substrate and exhibits regiospecificity for the C-2 hydroxyl group of the enediyne core mimic as the acceptor substrate. Remarkably, SgcC5 is also capable of catalyzing amide bond formation, albeit with significantly reduced efficiency, between (S)-3-chloro-5-hydroxy-β-tyrosyl-(S)-SgcC2 and (R)-2-amino-1-phenyl-1-ethanol, an alternative enediyne core mimic bearing an amine at its C-2 position. Thus, SgcC5 is capable of catalyzing both ester and amide bond formation, providing an evolutionary link between amide- and ester-forming condensation enzymes.

The Role of Tandem Acyl Carrier Protein Domains in Polyunsaturated Fatty Acid Biosynthesis

Acyl carrier protein (ACP) plays an essential role in fatty acid and polyketide biosynthesis, and most of the fatty acid synthases (FASs) and polyketide synthases (PKSs) known to date are characterized with a single ACP for each cycle of chain elongation. Polyunsaturated fatty acid (PUFA) biosynthesis is catalyzed by the PUFA synthase, and all PUFA synthases known to date contain tandem ACPs (ranging from 5 to 9). Using the Pfa PUFA synthase from Shewanella japonica as a model system, we report here that these tandem ACPs are functionally equivalent regardless of their physical location within the PUFA synthase subunit, but the total number of ACPs controls the overall PUFA titer. These findings set the stage to interrogate other domains and subunits of PUFA synthase for their roles in controlling the final PUFA products and could potentially be exploited to improve PUFA production.

An ATP-independent strategy for amide bond formation in antibiotic biosynthesis.

A-503083 B, a capuramycin-type antibiotic, contains an L-aminocaprolactam and an unsaturated hexuronic acid that are linked via an amide bond. A putative class C beta-lactamase (CapW) was identified within the biosynthetic gene cluster that-in contrast to the expected beta-lactamase activity-catalyzed an amide-ester exchange reaction to eliminate the L-aminocaprolactam with concomitant generation of a small but significant amount of the glyceryl ester derivative of A-503083 B, suggesting a potential role for an ester intermediate in the biosynthesis of capuramycins. A carboxyl methyltransferase, CapS, was subsequently demonstrated to function as an S-adenosylmethionine-dependent carboxyl methyltransferase to form the methyl ester derivative of A-503083 B. In the presence of free L-aminocaprolactam, CapW efficiently converts the methyl ester to A-503083 B, thereby generating a new amide bond. This ATP-independent amide bond formation using methyl esterification followed by an ester-amide exchange reaction represents an alternative to known strategies of amide bond formation.

Biosynthesis of the enediyne antitumor antibiotic C-1027 involves a new branching point in chorismate metabolism

C-1027 is an enediyne antitumor antibiotic composed of four distinct moieties: an enediyne core, a deoxy aminosugar, a β-amino acid, and a benzoxazolinate moiety. We now show that the benzoxazolinate moiety is derived from chorismate by the sequential action of two enzymes—SgcD, a 2-amino-2-deoxyisochorismate (ADIC) synthase and SgcG, an iron–sulfur, FMN-dependent ADIC dehydrogenase—to generate 3-enolpyruvoylanthranilate (OPA), a new intermediate in chorismate metabolism. The functional elucidation and catalytic properties of each enzyme are described, including spectroscopic characterization of the products and the development of a fluorescence-based assay for kinetic analysis. SgcD joins isochorismate (IC) synthase and 4-amino-4-deoxychorismate (ADC) synthase as anthranilate synthase component I (ASI) homologues that are devoid of pyruvate lyase activity inherent in ASI; yet, in contrast to IC and ADC synthase, SgcD has retained the ability to aminate chorismate identically to that observed for ASI. The net conversion of chorismate to OPA by the tandem action of SgcD and SgcG unambiguously establishes a new branching point in chorismate metabolism.

The Biosynthesis of Liposidomycin-like A-90289 Antibiotics Featuring a New Type of Sulfotransferase

Enzymes involved in peptidoglycan cell wall biosynthesis are proven targets for antibacterial drugs that have revolutionized medicine. Remarkably, however, the majority of the enzymes involved in peptidoglycan biosynthesis have yet to be exploited in the clinic. Bacterial translocase I (also annotated as MraY), which initiates the lipid cycle of peptidoglycan cell wall biosynthesis by transfer of phospho-N-acetylmuramic acid-pentapeptide from UDP-N-acetylmuramic acid-pentapeptide to undecaprenyl phosphate, represents one such enzyme for which there are no marketed antibiotic drugs. Given the emergence and re-emergence of drug-resistance pathogens, the essential nature of peptidoglycan in the viability of bacteria, and the lack of a bacterial translocase I activity in mammals, MraY is an attractive target for the design and development of new antibiotics. Recent efforts in several laboratories have revealed that several structurally diverse natural products potently inhibit bacterial translocase I. This includes several families of nucleoside antibiotics that have unique chemical scaffolds relative to clinically used antibiotics. The liposidomycins belong to one such family of nucleoside antibiotics called fatty acyl nucleosides that also includes the caprazamycins isolated from Streptomyces sp. MK730-62F2. The liposidomycins, the structure of which was initially reported in 1988, are characterized by four moieties, a 5’-C-glycyluridine, a 2’-sulfated aminoribose, a diazepanone, and a b-hydroxy fatty acid moiety of variable carbon chain length modified with an unusual 3-methylglutaryl group at the b-position. Caprazamycins, in contrast to liposidomycins, contain an additional permethylated rhamnose and lack the 2’-O-sulfate moiety. During screening for new compounds that inhibit bacterial translocase I, we isolated a series of related compounds from Streptomyces sp. SANK 60405 termed A-90289s that had properties characteristic of the fatty acyl nucleoside antibiotics including variable fatty acid side chains. NMR analysis—including DQF COSY, HMBC, and NOESY experiments—and MS analysis with collision-induced dissociation (CID) were consistent with A-90289A, consisting of a b-hydroxy palmitic acid moiety and having a structure identical to that of caprazamycin A but also containing a sulfate group characteristic of liposidomycins B-I and the other liposidomycins (Scheme 1). However, in contrast

Enzymatic Total Synthesis of Defucogilvocarcin M and Its Implications for Gilvocarcin Biosynthesis

Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Go 3592 and various other Streptomyces species. GV is usually produced along with its minor congeners, gilvocarcin M (3) and gilvocarcin E (5), that vary with respect to their side chain at the C8-position.[1] Several analogues of GV (for example, 6–8, Scheme 1), which are collectively called the gilvocarcin group of natural products, have been isolated from different Streptomyces species. All of these analogues contain the characteristic, polyketide-derived benzo[d]naphtho[1,2-b]pyran-6-one chromophore, but different C-glycosidically linked sugar units Scheme 1).[2] Members of this group of natural products are well-known for strong antitumor activities,[3] a unique mode of action,[4] and remarkably low toxicities.[2c, 5] However, the inherent poor solubility of these molecules appears to be a major obstacle in their development as therapeutics. The chemical syntheses that have been developed so far are unsuitable for generating a library of analogues,[6] whereas combinatorial biosynthetic efforts have shown more promise.[7] The continued advancement and successful implementation of such combinatorial biosynthetic and mutasynthetic approaches requires an in-depth knowledge of the biosynthetic pathway. Incorporation studies with isotope-labeled precursors[8] and genetic experiments[1a, 8d, 9] have revealed that the benzo[d]naphtho[1,2-b]pyran-6-one chromophore of the gilvocarcins is produced from a polyketide-derived angucyclinone intermediate through a complex oxidative rearrangement process. However, the details of the exact sequence of biosynthetic events and the enzymes that are involved have remained elusive. In this context, we herein report a complete, one-pot, enzymatic total synthesis of defucogilvocarcin M(1), a model compound that contains the unique chromophore common to all members of the gilvocarcin group of natural products.[10] The reconstitution of this pathway then enabled further investigation into the details of the oxidative rearrangement process of GV biosynthesis by systematic variation of the enzyme mixture used. For this approach we suggest the term “combinatorial biosynthetic enzymology”.

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5′-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O2, stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be Kmα-KG = 7.5 μm, KmUMP = 14 μm, and kcat ≈ 80 min−1. The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

Characterization of the Two-Component, FAD-Dependent Monooxygenase SgcC That Requires Carrier Protein-Tethered Substrates for the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027

C-1027 is a potent antitumor antibiotic composed of an apoprotein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the biosynthesis of which from l-alpha-tyrosine requires five proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth protein, SgcC5, catalyzes the incorporation of this beta-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (peptidyl carrier protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O 2 and FADH 2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted beta-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but does not accept 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. Together with the in vitro data for SgcC4, SgcC1, and SgcC3, the results establish that SgcC catalyzes the hydroxylation of ( S)-3-chloro-beta-tyrosyl-S-SgcC2 as the final step in the biosynthesis of the ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety prior to incorporation into C-1027. SgcC now represents the first biochemically characterized two-component, FAD-dependent monooxygenase that acts on a carrier-protein-tethered aromatic substrate.

Identification of the biosynthetic gene cluster of A-500359s in Streptomyces griseus SANK60196.

A-500359s, produced by Streptomyces griseus SANK60196, are inhibitors of bacterial phospho-N-acetylmuramyl-pentapeptide translocase. They are composed of three distinct moieties: a 5′-carbamoyl uridine, an unsaturated hexuronic acid and an aminocaprolactam. Two contiguous cosmids covering a 65-kb region of DNA and encoding 38 open reading frames (ORFs) putatively involved in the biosynthesis of A-500359s were identified. Reverse transcriptase PCR showed that most of the 38 ORFs are highly expressed during A-500359s production, but mutants that do not produce A-500359s did not express these same ORFs. Furthermore, orf21, encoding a putative aminoglycoside 3′-phosphotransferase, was heterologously expressed in Escherichia coli and Streptomyces albus, yielding strains having selective resistance against A-500359B, suggesting that ORF21 phosphorylates the unsaturated hexuronic acid as a mechanism of self-resistance to A-500359s. In total, the data suggest that the cloned region is involved in the resistance, regulation and biosynthesis of A-500359s.