Bacillus subtilis as a host for natural product discovery and engineering of biosynthetic gene clusters.

Covering: up to October 2023Many bioactive natural products are synthesized by microorganisms that are either difficult or impossible to cultivate under laboratory conditions, or that produce only small amounts of the desired compound. By transferring biosynthetic gene clusters (BGCs) into alternative host organisms that are more easily cultured and engineered, larger quantities can be obtained and new analogues with potentially improved biological activity or other desirable properties can be generated. Moreover, expression of cryptic BGCs in a suitable host can facilitate the identification and characterization of novel natural products. Heterologous expression therefore represents a valuable tool for natural product discovery and engineering as it allows the study and manipulation of their biosynthetic pathways in a controlled setting, enabling innovative applications. Bacillus is a genus of Gram-positive bacteria that is widely used in industrial biotechnology as a host for the production of proteins from diverse origins, including enzymes and vaccines. However, despite numerous successful examples, Bacillus species remain underexploited as heterologous hosts for the expression of natural product BGCs. Here, we review important advantages that Bacillus species offer as expression hosts, such as high secretion capacity, natural competence for DNA uptake, and the increasing availability of a wide range of genetic tools for gene expression and strain engineering. We evaluate different strain optimization strategies and other critical factors that have improved the success and efficiency of heterologous natural product biosynthesis in B. subtilis. Finally, future perspectives for using B. subtilis as a heterologous host are discussed, identifying research gaps and promising areas that require further exploration.

Exploring the selectivity of cytochrome P450 for enhanced novel anticancer agent synthesis.

Cytochrome P450 monooxygenases are an extensive and unique class of enzymes, which can regio- and stereo-selectively functionalise hydrocarbons by way of oxidation reactions. These enzymes are naturally occurring but have also been extensively applied in a synthesis context, where they are used as efficient biocatalysts. Recently, a biosynthetic pathway where a cytochrome P450 monooxygenase catalyses a critical step of the pathway was uncovered, leading to the production of a number of products that display high antitumour potency. In this work, we use computational techniques to gain insight into the factors that determine the relative yields of the different products. We use conformational search algorithms to understand the substrate stereochemistry. On a machine-learned 3D protein structure, we use molecular docking to obtain a library of favourable poses for substrate-protein interaction. With molecular dynamics, we investigate the most favourable poses for reactivity on a molecular level, allowing us to investigate which protein-substrate interactions favour a given product and thus gain insight into the product selectivity.

Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H+ -ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.

Beyond peptide bond formation: the versatile role of condensation domains in natural product biosynthesis.

Covering: up to the end of 2020Nonribosomal peptide synthetases are remarkable molecular machines that produce a wide range of structurally complex peptide natural products with important applications in medicine and agriculture. Condensation domains play a central role in these biosynthetic pathways by catalysing amide bond formation between various aminoacyl substrates. In recent years, however, it has become increasingly clear that the catalytic repertoire of C domains extends far beyond conventional peptide bond formation. C domains have been shown to perform highly diverse functions during nonribosomal peptide assembly, such as ß-lactam formation, dehydration, hydrolysis, chain length control, cycloaddition, Pictet-Spengler cyclization, Dieckmann condensation and recruitment of auxiliary enzymes. In this review, a comprehensive overview of the multifaceted role of C domains in the biosynthesis of specialized metabolites in bacteria and fungi is presented. Different perspectives are also offered on how the exceptional functional versatility of C domains may be exploited for bioengineering approaches to expand the chemical diversity of nonribosomal peptides and other natural products.

Genomics-Driven Discovery of a Novel Glutarimide Antibiotic from Burkholderia gladioli Reveals an Unusual Polyketide Synthase Chain Release Mechanism.

A gene cluster encoding a cryptic trans-acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI.

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

The zeamine antibiotics affect the integrity of bacterial membranes.

The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBDKZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of the OM rather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components.

A conserved motif flags acyl carrier proteins for ß-branching in polyketide synthesis.

Type I polyketide synthases often use programmed ß-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where ß-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with ß-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem ß-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting ß-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.

From competition to cure: the development of live biotherapeutic products for anticancer therapy in the iGEM competition.

Cancer is a leading cause of mortality globally, often diagnosed at advanced stages with metastases already present, complicating treatment efficacy. Traditional treatments like chemotherapy and radiotherapy face challenges such as lack of specificity and drug resistance. The hallmarks of cancer, as defined by Hanahan and Weinberg, describe tumors as complex entities capable of evolving traits that promote malignancy, including sustained proliferation, resistance to cell death, and metastasis. Emerging research highlights the significant role of the microbiome in cancer development and treatment, influencing tumor progression and immune responses. This review explores the potential of live biotherapeutic products (LBPs) for cancer diagnosis and therapy, focusing on projects from the International Genetically Engineered Machines (iGEM) competition that aim to innovate LBPs for cancer treatment. Analyzing 77 projects from 2022, we highlight the progress and ongoing challenges within this research field.

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Artificial intelligence for natural product drug discovery.

Developments in computational omics technologies have provided new means to access the hidden diversity of natural products, unearthing new potential for drug discovery. In parallel, artificial intelligence approaches such as machine learning have led to exciting developments in the computational drug design field, facilitating biological activity prediction and de novo drug design for molecular targets of interest. Here, we describe current and future synergies between these developments to effectively identify drug candidates from the plethora of molecules produced by nature. We also discuss how to address key challenges in realizing the potential of these synergies, such as the need for high-quality datasets to train deep learning algorithms and appropriate strategies for algorithm validation.

Total Synthesis of Kalimantacin A.

The kalimantacins make up a family of hybrid polyketide-nonribosomal peptide-derived natural products that display potent and selective antibiotic activity against multidrug resistant strains of Staphylococcus aureus. Herein, we report the first total synthesis of kalimantacin A, in which three fragments are prepared and then united via Sonogashira and amide couplings. The enantioselective synthetic approach is convergent, unlocking routes to further kalimantacins and analogues for structure-activity relationship studies and clinical evaluation.

A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis.

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues.

An unusual Burkholderia gladioli double chain-initiating nonribosomal peptide synthetase assembles 'fungal' icosalide antibiotics.

Burkholderia is a multi-talented genus of Gram-negative bacteria, which in recent years has become increasingly recognised as a promising source of bioactive natural products. Metabolite profiling of Burkholderia gladioli BCC0238 showed that it produces the asymmetric lipopeptidiolide antibiotic icosalide A1, originally isolated from a fungus. Comparative bioinformatics analysis of several genome-sequenced B. gladioli isolates identified a gene encoding a nonribosomal peptide synthase (NRPS) with an unusual architecture that was predicted to be responsible for icosalide biosynthesis. Inactivation of this gene in B. gladioli BCC0238 abolished icosalide production. PCR analysis and sequencing of total DNA from the original fungal icosalide A1 producer revealed it has a B. gladioli strain associated with it that harbours an NRPS with an identical architecture to that responsible for icosalide A1 assembly in B. gladioli BCC0238. Sequence analysis of the icosalide NRPS indicated that it contains two chain-initiating condensation (CI) domains. One of these is appended to the N-terminus of module 1 - a common architecture for NRPSs involved in lipopeptide assembly. The other is embedded in module 3, immediately downstream of a putative chain-elongating condensation domain. Analysis of the reactions catalysed by a tridomain construct from module 3 of the NRPS using intact protein mass spectrometry showed that the embedded CI domain initiates assembly of a second lipopeptide chain, providing key insights into the mechanism for asymmetric diolide assembly.

Pentamycin Biosynthesis in Philippine Streptomyces sp. S816: Cytochrome P450-Catalyzed Installation of the C-14 Hydroxyl Group.

Pentamycin is a polyene antibiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomoniasis, and mixed infections. Chemical instability has hindered its widespread application and development as a drug. Here, we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Genome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsible for filipin III biosynthesis. The ptnJ gene, which is absent from the filipin III biosynthetic gene cluster, was shown to encode a cytochrome P450 capable of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin biosynthesis, paving the way for biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsible for the assembly of filipin III and pentamycin, suggesting pentamycin production may be common in Streptomyces species.

Structural basis for chain release from the enacyloxin polyketide synthase.

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Elucidation of the relative and absolute stereochemistry of the kalimantacin/batumin antibiotics.

Kalimantacin A and batumin exhibit potent and selective antibiotic activity against Staphylococcus species including MRSA. Both compounds are formed via a hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS-NRPS) biosynthetic pathway and from comparison of the gene clusters it is apparent that batumin from Pseudomonas batumici and kalimantacin from P. fluorescens are the same compound. The linear structure of this unsaturated acid was assigned by spectroscopic methods, but the relative and absolute stereochemistry of the five stereocentres remained unknown. Herein we describe isolation of kalimantacin A and two further metabolites 17,19-diol 2 and 27-descarbomyl hydroxyketone 3 from cultures of P. fluorescens. Their absolute and relative stereochemistries are rigorously determined using a multidisciplinary approach combining natural product degradation and fragment synthesis with bioinformatics and NMR spectroscopy. Diol 2 has the 5R, 15S, 17S, 19R, 26R, 27R configuration and is the immediate biosynthetic precursor of the bioactive kalimantacin A formed by oxidation of the 17-alcohol to the ketone.

Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach.

Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo.