Precursor-directed Biosynthesis Research Articles

Precursor-Directed Biosynthesis of Panepoxydone Derivatives with Nitric Oxide Production Inhibitory Activity.

Panepoxydone is a natural NF-κB inhibitor isolated from basidiomycetes belonging to the genus Panus and Lentinus. It is biosynthesized from prenylhydroquinone through successive hydroxylation, epoxidation, and reduction reactions. In this study, we establish an efficient precursor-directed biosynthesis strategy for the structural expansion of panepoxydone based on its biosynthetic pathway. Supplementation of the panepoxydone-producing strain, Panus rudis, with various prenylhydroquinone analogues enabled the production of fourteen previously undescribed panepoxydone derivatives, panepoxyquinoid A-N (2-14). The obtained panepoxydone derivatives together with their parental molecules were evaluated for their inhibitory activity on LPS-induced NO production in RAW 264.7 cells. Compounds 1, 5-6, 10-11, and 14-15 displayed significant suppressive effects on LPS-induced NO production with IC50 values ranging from 4.3 to 30.1 μM.

Exploring the Biosynthetic Potential of Tistrella Species for Producing Didemnin Antitumor Agents.

Didemnins are a class of cyclic depsipeptides derived from sea tunicates that exhibit potent anticancer, antiviral, and immunosuppressive properties. Although certain Tistrella species can produce didemnins, their complete biosynthetic potential remains largely unexplored. In this study, we utilize feature-based molecular networking to analyze the metabolomics of Tistrella mobilis and Tistrella bauzanensis, focusing on the production of didemnin natural products. In addition to didemnin B, we identify nordidemnin B and [hysp2]didemnin B, as well as several minor didemnin analogs. Heterologous expression of the didemnin biosynthetic gene cluster in a Streptomyces host results in the production of only didemnin B and nordidemnin B in limited quantities. Isotope-labeling studies reveal that the substrate promiscuity of the adenylation domains during biosynthesis leads to the accumulation of nordidemnin B and [hysp2]didemnin B. Additionally, precursor-directed biosynthesis is applied to generate eight novel didemnin derivatives by supplementing the culture with structurally related amino acids. Furthermore, we increased the titers of nordidemnin B and [hysp2]didemnin B by supplementing the fermentation medium with l-valine and l-isoleucine, respectively. Finally, both compounds undergo side-chain oxidation to enhance their biological activity, with their anticancer properties found to be as potent as plitidepsin.

Harnessing precursor-directed biosynthesis with glucose derivatives to access cotton fibers with enhanced physical properties

Cotton ovule invitro cultures are a promising platform for exploring biofabrication of fibers with tailored properties. When the ovules' growth medium is supplemented with chemically synthesized cellulose precursors, it results in their integration into the developing fibers, thereby tailoring their end properties. Here, we report the feeding of synthetic glucosyl phosphate derivative, 6-deoxy-6-fluoro-glucose-1-phosphate (6F-Glc-1P) to cotton ovules growing invitro, demonstrating the metabolic incorporation of 6F-Glc into the fibers with enhanced mechanical properties and moisture-retention capacity while emphasizing the role of molecular hierarchical architecture in defining functional characteristics and mechanical properties. This incorporation strategy bypasses the early steps of conventional metabolic pathways while broadening the range of functionalities that can be employed to customize fiber end properties. Our approach combines materials science, chemistry, and plant sciences to illustrate the innovation required to find alternative solutions for sustainable production of functional cotton fibers with enhanced and emergent properties.

Precursor-Directed Biosynthesis of Neoantimycin Derivatives with Selective Cytotoxicity.

A precursor-directed biosynthesis approach led to the accumulation of seven new neoantimycin derivatives (1-7) from Streptomyces conglobatus RJ2. Structure elucidation was conducted using NMR and HRESIMS analysis, and the absolute configuration was determined by advanced Marfey's method, Mosher's analysis, and ECD analysis. The obtained compounds revealed selective and significant cytotoxicity, specifically against colorectal cancer cells bearing the K-ras mutation, with IC50 values ranging from 40 nM to 3.5 μM.

WLIP, WLIPβ, and WLIPγ Produced from Pseudomonas canadensis Q3-1 via Precursor-Directed Biosynthesis and Their Roles on Biocontrol of Phytophthora Blight in Peppers.

White line-inducing principle (WLIP, 1), together with two new cyclic lipopeptides (CLPs) WLIPβ (2) and WLIPγ (3), were characterized from the supernatant of Pseudomonas canadensis Q3-1 via precursor-directed biosynthesis (PDB) in the current study. They were purified from the supernatant of P. canadensis Q3-1 by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC), and their structures were mainly determined via bioinformatic analyses, spectrometric and spectroscopic techniques, as well as single crystal X-ray diffraction (XRD). These WLIPs share (R)-3-hydroxydecanoic acid (HDA), but they differ from each other in the composition of peptidic sequences. In addition, these CLPs showed biocontrol activities against Phytophthora blight (caused by Phytophthora capsici) in peppers. Collectively, this study has shown that PDB could be used for generating new CLPs in Pseudomonas spp. Moreover, we have confirmed that WLIP, WLIPβ, and WLIPγ could be used as lead agrochemicals to control Phytophthora blight in peppers.

Precursor-Directed Biosynthesis of Antialgal Fluorinated Bacillamide Derivatives in Bacillus atrophaeus.

The bacillamides are a class of indole alkaloids produced by the Bacillus genus that possess significant antialgal activity. Incorporation of fluorine into the bacillamides was carried out using a precursor-directed biosynthesis approach, with 4-, 5-, and 6-fluorotryptophan added to growing cultures of Bacillus atrophaeus IMG-11. This yielded the corresponding fluorinated analogues of bacillamides A and C, in addition to new derivatives of the related metabolite N-acetyltryptamine, thus demonstrating a degree of plasticity in the bacillamide biosynthetic pathway. The bacillamide derivatives were tested for activity against bloom-forming algae, which revealed that fluorination could improve the antialgal activity of these compounds in a site-specific manner, with fluorination at the 6-position consistently resulting in improved activity.

Precursor-directed biosynthesis and biological activity of tripropeptin Cpip, a new tripropeptin C analog containing pipecolic acid.

Tripropeptin C, a non-ribosomal cyclic lipopeptide containing three proline residues, exhibits excellent efficacy in the mouse-methicillin-resistant Staphylococcus aureus septicemia model. Since tripropeptins contain L-prolyl-D-proline and, as a result, are known to form a hairpin structure in proteins, it was of interest to determine whether this substructure contributes to their antibacterial activity. In this study, prolines in tripropeptin C were replaced with pipecolic acid(s) using precursor-directed biosynthesis. Only a new tripropeptin analog, tripropeptin Cpip, which has one L-pipecolic acid in place of L-proline, was isolated. The in vitro antimicrobial activity of the new analog was approximately two to four times weaker activity against Gram-positive bacteria, including drug-resistant species, compared with that of tripropeptin C. These results suggest that the L-prolyl-D-proline substructure plays an important role in the observed potency of tripropeptins.

Sulfarulumines A-J, sulfa-containing talaroenamines with acetylcholinesterase inhibitory activity from Penicillium malacosphaerulum

A one-pot/two-stage precursor-directed biosynthesis approach was used to introduce selected sulfanilamide derivatives into the talaroenamine pathway in a Yellow River wetland-derived fungus. During this approach, precursors p-methylaniline and sulfanilamide derivatives were added to the medium in two stages, improving the substrate acceptance of biosynthetic intermediate cyclohexanedione to form diverse talaroenamines. Ten new sulfa-containing talaroenamine derivatives, sulfarulumines A-J (1–10), were isolated and structurally characterized. Compound 2 displayed acetylcholinesterase (AChE) inhibitory effect with an IC50 value of 4.9 µM. Molecular docking study suggested that 2 exerted inhibitory activity by binding to an active site of the enzyme.

Dynemicin A Derivatives as Potential Cancer Chemotherapeutics by Mutasynthesis.

The enediyne antitumor antibiotics have remarkable structures and exhibit potent DNA cleavage properties that have inspired continued interest as cancer therapeutics. Their complex structures and high reactivity, however, pose formidable challenges to their production and development in the clinic. We report here proof-of-concept studies using a mutasynthesis strategy to combine chemical synthesis of select modifications to a key iodoanthracene-γ-thiolactone intermediate in the biosynthesis of dynemicin A and all other known anthraquinone-fused enediynes (AFEs). By chemical complementation of a mutant bacterial producer that is incapable of synthesizing this essential building block, we show that derivatives of dynemicin can be prepared substituted in the A-ring of the anthraquinone motif. In the absence of competition from native production of this intermediate, the most efficient utilization of these externally-supplied structural analogues for precursor-directed biosynthesis becomes possible. To achieve this goal, we describe the required Δorf15 blocked mutant and a general synthetic route to a library of iodoanthracene structural variants. Their successful incorporation opens the door to enhancing DNA binding and tuning the bioreductive activation of the modified enediynes for DNA cleavage.

Precursor-Directed Biosynthesis and Biological Testing of omega-Alicyclic- and neo-Branched Tunicamycin N-Acyl Variants.

Tunicamycins (TUNs) are Streptomyces-derived natural products, widely used to block protein N-glycosylation in eukaryotes or cell wall biosynthesis in bacteria. Modified or synthetic TUN analogues that uncouple these activities have considerable potential as novel mode-of-action antibacterial agents. Chemically modified TUNs reported previously with attenuated activity on yeast have pinpointed eukaryotic-specific chemophores in the uridyl group and the N-acyl chain length and terminal branching pattern. A small molecule screen of fatty acid biosynthetic primers identified several novel alicyclic- and neo-branched TUN N-acyl variants, with primer incorporation at the terminal omega-acyl position. TUNs with unique 5- and 6-carbon ω-cycloalkane and ω-cycloalkene acyl chains are produced under fermentation and in yields comparable with the native TUN. The purification, structural assignments, and the comparable antimicrobial properties of 15 of these compounds are reported, greatly extending the structural diversity of this class of compounds for potential medicinal and agricultural applications.

Insecticidal Cyclodepsitetrapeptides from Mortierella alpina.

Early diverging fungi, such as Mortierella alpina, are an emerging source of bioactive peptides. By screening 22 fungal isolates together with precursor-directed biosynthesis, a family of threonine-linked cyclotetradepsipeptides, the cycloacetamides A-F (1-6), was identified. The structure elucidation was conducted using NMR and HR-ESI-MS/MS analyses, and the absolute configuration was determined by Marfey's analysis and total synthesis. Cycloacetamides are not cytotoxic to human cells, while being highly selectively insecticidal against fruit fly larvae.

Engineering Siderophore Biosynthesis and Regulation Pathways to Increase Diversity and Availability.

Siderophores are small metal chelators synthesized by numerous organisms to access iron. These secondary metabolites are ubiquitously present on Earth, and because their production represents the main strategy to assimilate iron, they play an important role in both positive and negative interactions between organisms. In addition, siderophores are used in biotechnology for diverse applications in medicine, agriculture and the environment. The generation of non-natural siderophore analogs provides a new opportunity to create new-to-nature chelating biomolecules that can offer new properties to expand applications. This review summarizes the main strategies of combinatorial biosynthesis that have been used to generate siderophore analogs. We first provide a brief overview of siderophore biosynthesis, followed by a description of the strategies, namely, precursor-directed biosynthesis, the design of synthetic or heterologous pathways and enzyme engineering, used in siderophore biosynthetic pathways to create diversity. In addition, this review highlights the engineering strategies that have been used to improve the production of siderophores by cells to facilitate their downstream utilization.

Biosynthesis of Tetronates by a Nonribosomal Peptide Synthetase-Polyketide Synthase System.

A cryptic tetronate biosynthetic pathway was identified in Kitasatospora niigatensis DSM 44781 via heterologous expression. Distinct from the currently known biosynthetic pathways, this system utilizes a partially functional nonribosomal peptide synthetase and a broadly selective polyketide synthase to direct the assembly and lactonization of the tetronate scaffold. By employing a permissive crotonyl-CoA reductase/carboxylase to provide different extender units, seven new tetronates (kitaniitetronins A-G) were obtained via precursor-directed biosynthesis.

Biosynthetic diversification of non-ribosomal peptides through activity-based protein profiling of adenylation domains.

We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.

Biosynthesis of rumbrins and inspiration for discovery of HIV inhibitors

Investigation on how nature produces natural compounds with chemical and biological diversity at the genetic level offers inspiration for the discovery of new natural products and even their biological targets. The polyketide rumbrin (1) is a lipid peroxide production and calcium accumulation inhibitor, which contains a chlorinated pyrrole moiety that is a rare chemical feature in fungal natural products. Here, we identify the biosynthetic gene cluster (BGC) rum of 1 and its isomer 12E-rumbrin (2) from Auxarthron umbrinum DSM3193, and elucidate their biosynthetic pathway based on heterologous expression, chemical complementation, and isotopic labeling. We show that rumbrins are assembled by a highly reducing polyketide synthase (HRPKS) that uniquely incorporates a proline-derived pyrrolyl-CoA starer unit, and followed by methylation and chlorination. Sequent precursor-directed biosynthesis was able to yield a group of rumbrin analogues. Remarkably, inspired by the presence of a human immunodeficiency virus (HIV)-Nef-associated gene in the rum cluster, we predicted and pharmacologically demonstrated rumbrins as potent inhibitors of HIV at the nanomolar level. This work enriches the recognition of unconventional starter units of fungal PKSs and provides a new strategy for genome mining-guided drug discovery.

Production of Multiple Talaroenamines from Penicillium malacosphaerulum via One-Pot/Two-Stage Precursor-Directed Biosynthesis.

Nineteen new talaroenamine derivatives, talaroenamines F1-F19 (1-19), were isolated from the Yellow River wetland derived Penicillium malacosphaerulum HPU-J01 by use of a one-pot/two-stage precursor-directed biosynthesis approach. During this approach, the initial precursor p-methylaniline was first used as a carrier to capture the biologically synthesized cyclohexanedione to produce talaroenamine F, and then the other aniline derivatives were employed to replace the p-methylaniline fragment of talaroenamine F to generate the final products. LC-MS analysis showed that only four compounds (2, 8, 10, and 12) could be produced by the traditional precursor-directed biosynthesis in which the aniline precursors were added simultaneously. Compound 14 was cytotoxic against the K562 cell line with an IC50 value of 2.2 μM. This work demonstrated the one-pot/two-stage precursor-directed biosynthesis could improve substrate acceptance leading to the production of diverse talaroenamines.

Alternative Benzoxazole Assembly Discovered in Anaerobic Bacteria Provides Access to Privileged Heterocyclic Scaffold.

Benzoxazole scaffolds feature prominently in diverse synthetic and natural product‐derived pharmaceuticals. Our understanding of their bacterial biosynthesis is, however, limited to ortho‐substituted heterocycles from actinomycetes. We report an overlooked biosynthetic pathway in anaerobic bacteria (typified in Clostridium cavendishii) that expands the benzoxazole chemical space to meta‐substituted heterocycles and heralds a distribution beyond Actinobacteria. The first benzoxazoles from the anaerobic realm (closoxazole A and B) were elucidated by NMR and chemical synthesis. By genome editing in the native producer, heterologous expression in Escherichia coli, and systematic pathway dissection we show that closoxazole biosynthesis invokes an unprecedented precursor usage (3‐amino‐4‐hydroxybenzoate) and manner of assembly. Synthetic utility was demonstrated by the precursor‐directed biosynthesis of a tafamidis analogue. A bioinformatic survey reveals the pervasiveness of related gene clusters in diverse bacterial phyla.

Carbon-nitrogen bond formation to construct novel polyketide-indole hybrids from the indole-3-carbinol exposed culture of Daldinia eschscholzii

A plenty of cytochrome P450s have been annotated in the Daldinia eschosholzii genome. Inspired by the fact that some P450s have been reported to catalyze the carbon-nitrogen (C–N) bond formation, we were curious about whether hybrids through C–N bond formation could be generated in the indole-3-carbinol (I3C) exposed culture of D. eschscholzii. As expected, two skeletally undescribed polyketide-indole hybrids, designated as indolpolyketone A and B (1 and 2), were isolated and assigned to be constructed through C–N bond formation. Their structures were elucidated by 1D and 2D NMR spectra. The absolute configurations of 1 and 2 were determined by comparing the recorded and calculated electronic circular dichroism (ECD) spectra. Furthermore, the plausible biosynthetic pathways for 1 and 2 were proposed. Compounds 1 and 2 exhibited significant antiviral activity against H1N1 with IC50 values of 45.2 and 31.4 μM, respectively. In brief, compounds 1 and 2 were reported here for the first time and were the first example of polyketide-indole hybrids pieced together through C–N bond formation in the I3C-exposed culture of D. eschscholzii. Therefore, this study expands the knowledge about the chemical production of D. eschscholzii through precursor-directed biosynthesis (PDB).

Neochrysosporazines: Precursor-Directed Biosynthesis Defines a Marine-Derived Fungal Natural Product P-Glycoprotein Inhibitory Pharmacophore.

Upregulation of ATP binding cassette (ABC) transporter efflux pumps (i.e. P-glycoprotein, P-gp) can impart multidrug resistance, rendering many chemotherapeutics ineffective and seriously limiting treatment regimes. While ABC transporters remain an attractive target for therapeutic intervention, the development of clinically useful small-molecule inhibitors has proved challenging. In this report, we describe the structure-activity relationship (SAR) analysis of a newly discovered P-gp inhibitory pharmacophore, phenylpropanoid piperazine chrysosporazines, produced by co-isolated marine-derived fungi. In the absence of any total syntheses, we apply an innovative precursor-directed biosynthesis strategy that successfully repurposed fungal biosynthetic output, allowing us to isolate, characterize, and identify the structurally diverse neochrysosporazines A-Q. SAR analysis utilizing all known (and new) neochrysosporazines, chrysosporazines, and azachrysosporazines, plus semi-synthetic analogues, established the key structure requirements for the P-gp inhibitory pharmacophore, and, in addition, identified non-essential sites that allow for the design of affinity and other conjugated probes.

Macrophage-targeting oligopeptides from Mortierella alpina.

The realm of natural products of early diverging fungi such as Mortierella species is largely unexplored. Herein, the nonribosomal peptide synthetase (NRPS) MalA catalysing the biosynthesis of the surface-active biosurfactants, malpinins, has been identified and biochemically characterised. The investigation of the substrate specificity of respective adenylation (A) domains indicated a substrate-tolerant enzyme with an unusual, inactive C-terminal NRPS module. Specificity-based precursor-directed biosynthesis yielded 20 new congeners produced by a single enzyme. Moreover, MalA incorporates artificial, click-functionalised amino acids which allowed postbiosynthetic coupling to a fluorophore. The fluorescent malpinin conjugate penetrates mammalian cell membranes via an phagocytosis-mediated mechanism, suggesting Mortierella oligopeptides as carrier peptides for directed cell targeting. The current study demonstrates substrate-specificity testing as a powerful tool to identify flexible NRPS modules and highlights basal fungi as reservoir for chemically tractable compounds in pharmaceutical applications.