Complete Biosynthetic Pathway of the Phosphonate Phosphonothrixin: Two Distinct Thiamine Diphosphate-Dependent Enzymes Divide the Work to Form a C-C Bond.

Phosphonates often exhibit biological activities by mimicking the phosphates and carboxylates of biological molecules. The phosphonate phosphonothrixin (PTX), produced by the soil-dwelling bacterium Saccharothrix sp. ST-888, exhibits herbicidal activity. In this study, we propose a complete biosynthetic pathway for PTX by reconstituting its biosynthesis in vitro. Our intensive analysis demonstrated that two dehydrogenases together reduce phosphonopyruvate (PnPy) to 2-hydroxy-3-phosphonopropanoic acid (HPPA) to accelerate the thermodynamically unfavorable rearrangement of phosphoenolpyruvate (PEP) to PnPy. The next four enzymes convert HPPA to (3-hydroxy-2-oxopropyl)phosphonic acid (HOPA). In the final stage of PTX biosynthesis, the "split-gene" transketolase homologue, PtxB5/6, catalyzes the transfer of a two-carbon unit attached to the thiamine diphosphate (TPP) cofactor (provided by the acetohydroxyacid synthase homologue, PtxB7) to HOPA to produce PTX. This study reveals a unique C-C bond formation in which two distinct TPP-dependent enzymes, PtxB5/6 and PtxB7, divide the work to transfer an acetyl group, highlighting an unprecedented biosynthetic strategy for natural products.

Total Synthesis and Stereochemistry Assignment of Nucleoside Antibiotic A-94964.

A collective total synthesis of eight diastereoisomers associated with NMR analysis leads to a full stereochemistry assignment of the structurally unique nucleoside antibiotic A-94964, which features an octuronic acid uridine core decorated with an α-D-mannopyranosyl residue and an α-D-N-acylglucosaminopyranosyl residue via a phosphodiester bridge.

Biosynthetic pathways and enzymes involved in the production of phosphonic acid natural products.

Phosphonates are organophosphorus compounds possessing a characteristic C-P bond in which phosphorus is directly bonded to carbon. As phosphonates mimic the phosphates and carboxylates of biological molecules to potentially inhibit metabolic enzymes, they could be lead compounds for the development of a variety of drugs. Fosfomycin (FM) is a representative phosphonate natural product that is widely used as an antibacterial drug. Here, we review the biosynthesis of FM, which includes a recent breakthrough to find a missing link in the biosynthetic pathway that had been a mystery for a quarter-century. In addition, we describe the genome mining of phosphonate natural products using the biosynthetic gene encoding an enzyme that catalyzes C-P bond formation. We also introduce the chemoenzymatic synthesis of phosphonate derivatives. These studies expand the repertoires of phosphonates and the related biosynthetic machinery. This review mainly covers the years 2012-2020.

Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in ficellomycin essential for its biological activity.

The 1-azabicyclo[3.1.0]hexane ring is a key moiety in natural products for biological activities against bacteria, fungi, and tumor through DNA alkylation. Ficellomycin is a dipeptide that consists of l-valine and a non-proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure. Although the biosynthetic gene cluster of ficellomycin has been identified, the biosynthetic pathway currently remains unclear. We herein report the final stage of ficellomycin biosynthesis involving ring modifications and successive dipeptide formation. After the ring is formed, the hydroxy group of the ring is converted into the guanidyl unit by three enzymes, which include an aminotransferase with a novel inter ω-ω amino-transferring activity. In the last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid is connected with l-valine by an amino acid ligase to yield ficellomycin. The present study revealed a new machinery that expands the structural and biological diversities of natural products.

The Amipurimycin and Miharamycin Biosynthetic Gene Clusters: Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucleoside Antibiotics.

Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (-)-cispentacin and N5-hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny. Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimycin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the biosynthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which, along with labeled precursor feeding studies, implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N5-hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (-)-cispentacin.

Biosynthesis of the uridine-derived nucleoside antibiotic A-94964: identification and characterization of the biosynthetic gene cluster provide insight into the biosynthetic pathway.

The natural product A-94964 is a uridine-derived nucleoside antibiotic isolated from Streptomyces sp. SANK 60404. In this study, we propose a biosynthetic pathway for A-94964 using gene deletion experiments coupled with in silico analysis of the biosynthetic gene cluster. This study provides insights into the unique biosynthetic pathway for A-94964.

Amino-group carrier-protein-mediated secondary metabolite biosynthesis in Streptomyces.

Amino-group carrier proteins (AmCPs) mediate the biosynthesis of lysine and arginine in some bacteria and archaea. Here we demonstrate that an uncharacterized AmCP-mediated biosynthetic system functions to biosynthesize the previously uncharacterized and nonproteinogenic amino acid (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH) in Streptomyces sp. SANK 60404. DADH is incorporated into a novel peptide metabolite, vazabitide A, featuring an azabicyclo-ring structure, by nonribosomal peptide synthetases and successive modification enzymes in this bacterium. As the AmCP-mediated machinery for DADH biosynthesis is widely distributed in bacteria, further analysis of uncharacterized AmCP-containing gene clusters will lead to the discovery of novel bioactive compounds and novel biosynthetic enzymes.

Biosynthesis of Trehangelin in Polymorphospora rubra K07-0510: Identification of Metabolic Pathway to Angelyl-CoA.

Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07-0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase-like protein (thgC), α-amylase (thgD), 3-ketoacyl-ACP synthase III (thgI), 3-ketoacyl-ACP reductase (thgK), enoyl-CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl-CoA and methylmalonyl-CoA to 2-methylacetoacetyl-CoA (MAA-CoA), ThgK catalyses NADPH-dependent reduction of MAA-CoA to 3-hydroxy-2-methylbutyryl-CoA (HMB-CoA) and ThgH catalyses the dehydration of HMB-CoA to angelyl-CoA (AN-CoA). This is the first report on the elucidation of the enzymatic formation of AN-CoA.

Discovery of type II polyketide synthase-like enzymes for the biosynthesis of cispentacin.

Type II polyketide synthases (PKSs) normally synthesize polycyclic aromatic compounds in nature, and the potential to elaborate further diverse skeletons was recently revealed by the discovery of a polyene subgroup. Here, we show a type II PKS machinery for the biosynthesis of a five-membered nonaromatic skeleton contained in the nonproteinogenic amino acid cispentacin and the plant toxin coronatine. We successfully produce cispentacin in a heterologous host and reconstruct its biosynthesis using seven recombinant proteins in vitro. Biochemical analyses of each protein reveal the unique enzymatic reactions, indicating that a heterodimer of type II PKS-like enzymes (AmcF-AmcG) catalyzes a single C2 elongation as well as a subsequent cyclization on the acyl carrier protein (AmcB) to form a key intermediate with a five-membered ring. The subsequent reactions, which are catalyzed by a collection of type II PKS-like enzymes, are also peculiar. This work further expands the definition of type II PKS and illuminates an unexplored genetic resource for natural products.

Frequency and component analysis of contaminants generated in preparation of anticancer agents using closed system drug transfer devices (CSTDs).

Occupational exposure of anticancer agents during their preparation has been recognized as a serious hazard. Closed system drug transfer devices (CSTDs) enable "safe" preparation of agents for medical personnel and ensure a safe hospital environment. However, artificial particles of infusion materials have been reported during CSTD use. Here, the incidence of insoluble fine particles during preparation of anticancer agents using CSTDs was examined. Visible insoluble fine particles were found in 465 (9.4%) of 4948 treatment cases at Ehime University Hospital with CSTD use. Contaminants occurred more frequently during preparation of monoclonal antibodies than cytotoxic anticancer agents (19.4% vs. 4.1%, respectively, P < 0.01). A similar survey was conducted at nine hospitals to investigate the incidence of insoluble fine particles with or without CSTDs. Insoluble fine particles were detected in 113 (15.4%) of 732 treatment cases during preparation of monoclonal antibodies with CSTD use. In contrast, the occurrence of insoluble fine particles without CSTDs was found in only 3 (0.073%) of 4113 treatment cases. Contamination with CSTDs might cause harmful effects on patients during cancer therapy. We strongly recommend the use of in-line filters combined with infusion routes after CSTD use to avoid contamination-associated adverse events.

Biosynthesis of the nucleoside antibiotic angustmycins: identification and characterization of the biosynthetic gene cluster reveal unprecedented dehydratase required for exo-glycal formation.

The nucleoside antibiotic angustmycin, produced by some Streptomyces strains, is composed of adenine and C6 sugar and shows antibiotic and antitumor activities. In this study, we propose a biosynthetic pathway for angustmycin using a heterologous expression experiment coupled with in silico analysis of the angustmycin biosynthetic gene (agm) cluster. The biochemical characterization of Agm6 demonstrated its role in angustmycin biosynthesis as an unprecedented dehydratase.

Recent advances in the biosynthesis of nucleoside antibiotics.

Nucleoside antibiotics are a diverse class of natural products with promising biomedical activities. These compounds contain a saccharide core and a nucleobase. Despite the large number of nucleoside antibiotics that have been reported, biosynthetic studies on these compounds have been limited compared with those on other types of natural products such as polyketides, peptides, and terpenoids. Due to recent advances in genome sequencing technology, the biosynthesis of nucleoside antibiotics has rapidly been clarified. This review covering 2009-2019 focuses on recent advances in the biosynthesis of nucleoside antibiotics.

Identification and heterologous expression of the actinoallolide biosynthetic gene cluster.

Actinoallolides are anti-trypanosomal macrolides isolated from the secondary metabolites of two endophytic actinomycete strains, Actinoallomurus fulvus MK10-036 and K09-0307. A putative actinoallolide biosynthetic gene cluster was predicted from the genome sequence of the strain K09-0307. The gene cluster spans a contiguous 53 kb DNA region that comprises seven genes encoding three PKSs (aalA1, aalA2, and aalA3), cytochrome P450 (aalB), acyl-CoA dehydrogenase (aalC), crotonyl-CoA reductase (aalD), and TetR family regulator (aalR). The entire gene cluster was cloned into a plasmid pYIK1 by assembling DNA fragments, which were obtained from two cosmids containing left and right parts of the gene cluster. Following the introduction of an ermE* promoter at 100bp upstream from the start codon of aalA1, the gene cluster was introduced into Streptomyces coelicolor M1152. Subsequent LC-MS analysis revealed production of actinoallolide A in the culture broth. Thus, the actinoallolide biosynthetic gene cluster was identified by heterologous expression in Streptomyces.

Fosfomycin Biosynthesis via Transient Cytidylylation of 2-Hydroxyethylphosphonate by the Bifunctional Fom1 Enzyme.

Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, etc. Its biosynthesis starts with the formation of a carbon-phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 N-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 C-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism via such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family.

Efficient anti-tumor effect of photodynamic treatment with polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer encapsulating hydrophobic porphyrin derivative.

To develop potent and safer formulation of photosensitizer for cancer photodynamic therapy (PDT), we tried to formulate hydrophobic porphyrin derivative, photoprotoporphyrin IX dimethyl ester (PppIX-DME), into polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer (PN-Por). The mean particle size of PN-Por prepared was around 80nm and the zeta potential was determined to be weakly negative. In vitro phototoxicity study for PN-Por clearly indicated the significant phototoxicity of PN-Por for three types of tumor cells tested (Colon-26 carcinoma (C26), B16BL6 melanoma and Lewis lung cancer cells) in the PppIX-DME concentration-dependent fashion. Furthermore, it was suggested that the release of PppIX-DME from PN-Por would gradually occur to provide the sustained release of PppIX-DME. In vivo pharmacokinetics of PN-Por after intravenous administration was evaluated in C26 tumor-bearing mice, and PN-Por exhibited low affinity to the liver and spleen and was therefore retained in the blood circulation for a long time, leading to the efficient tumor disposition of PN-Por. Furthermore, significant and highly effective anti-tumor effect was confirmed in C26 tumor-bearing mice with the local light irradiation onto C26 tumor tissues after PN-Por injection. These findings indicate the potency of PN-Por for the development of more efficient PDT-based cancer treatments.