ABC-HuMi: the Atlas of Biosynthetic Gene Clusters in the Human Microbiome.

The human microbiome has emerged as a rich source of diverse and bioactive natural products, harboring immense potential for therapeutic applications. To facilitate systematic exploration and analysis of its biosynthetic landscape, we present ABC-HuMi: the Atlas of Biosynthetic Gene Clusters (BGCs) in the Human Microbiome. ABC-HuMi integrates data from major human microbiome sequence databases and provides an expansive repository of BGCs compared to the limited coverage offered by existing resources. Employing state-of-the-art BGC prediction and analysis tools, our database ensures accurate annotation and enhanced prediction capabilities. ABC-HuMi empowers researchers with advanced browsing, filtering, and search functionality, enabling efficient exploration of the resource. At present, ABC-HuMi boasts a catalog of 19 218 representative BGCs derived from the human gut, oral, skin, respiratory and urogenital systems. By capturing the intricate biosynthetic potential across diverse human body sites, our database fosters profound insights into the molecular repertoire encoded within the human microbiome and offers a comprehensive resource for the discovery and characterization of novel bioactive compounds. The database is freely accessible at https://www.ccb.uni-saarland.de/abc_humi/.

Investigation of the Odilorhabdin Biosynthetic Gene Cluster Using NRPS Engineering.

The recently identified natural product NOSO-95A from entomopathogenic Xenorhabdus bacteria, derived from a biosynthetic gene cluster (BGC) encoding a non-ribosomal peptide synthetase (NRPS), was the first member of the odilorhabdin class of antibiotics. This class exhibits broad-spectrum antibiotic activity and inspired the development of the synthetic derivative NOSO-502, which holds potential as a new clinical drug by breaking antibiotic resistance. While the mode of action of odilorhabdins was broadly investigated, their biosynthesis pathway remained poorly understood. Here we describe the heterologous production of NOSO-95A in Escherichia coli after refactoring the complete BGC. Since the production titer was low, NRPS engineering was applied to uncover the underlying biosynthetic principles. For this, modules of the odilorhabdin NRPS fused to other synthetases were co-expressed with candidate hydroxylases encoded in the BGC allowing the characterization of the biosynthesis of three unusual amino acids and leading to the identification of a prodrug-activation mechanism by deacylation. Our work demonstrates the application of NRPS engineering as a blueprint to mechanistically elucidate large or toxic NRPS and provides the basis to generate novel odilorhabdin analogues with improved properties in the future.

Genome Mining Enabled by Biosynthetic Characterization Uncovers a Class of Benzoxazolinate-Containing Natural Products in Diverse Bacteria.

Benzoxazolinate is a rare bis-heterocyclic moiety that interacts with proteins and DNA and confers extraordinary bioactivities on natural products, such as C-1027. However, the biosynthetic gene responsible for the key cyclization step of benzoxazolinate remains unclear. Herein, we show a putative acyl AMP-ligase responsible for the last cyclization step. We used the enzyme as a probe for genome mining and discovered that the orphan benzobactin gene cluster in entomopathogenic bacteria prevails across Proteobacteria and Firmicutes. It turns out that Pseudomonas chlororaphis produces various benzobactins, whose biosynthesis is highlighted by a synergistic effect of two unclustered genes encoding enzymes on boosting benzobactin production; the formation of non-proteinogenic 2-hydroxymethylserine by a serine hydroxymethyltransferase; and the types I and II NRPS architecture for structural diversity. Our findings reveal the biosynthetic potential of a widespread benzobactin gene cluster.

Natural Products from Photorhabdus and Other Entomopathogenic Bacteria.

Although the first natural products (NP) from Photorhabdus and Xenorhabdus bacteria have been known now for almost 30 years, a huge variety of new compounds have been identified in the last 5-10 years, mainly due to the application of modern mass spectrometry. Additionally, application of molecular methods that allow the activation of NP production in several different strains as well as efficient heterologous expression methods have led to the production and validation of many new compounds. In this chapter we discuss the benefit of using Photorhabdus as a model system for microbial chemical ecology. We also examine non-ribosomal peptide synthetases as the most important pathway for NP production. Finally, we discuss the origin and function of all currently known NPs and the development of the molecular and chemical tools used to identify these NPs faster.

A multifunctional enzyme is involved in bacterial ether lipid biosynthesis.

Fatty acid-derived ether lipids are present not only in most vertebrates but also in some bacteria. Here we describe what is to our knowledge the first gene cluster involved in the biosynthesis of such lipids in myxobacteria that encodes the multifunctional enzyme ElbD, which shows similarity to polyketide synthases. Initial characterization of elbD mutants in Myxococcus xanthus and Stigmatella aurantiaca showed the importance of these ether lipids for fruiting body formation and sporulation.

A Photorhabdus natural product inhibits insect juvenile hormone epoxide hydrolase.

Simple urea compounds ("phurealipids") have been identified from the entomopathogenic bacterium Photorhabdus luminescens, and their biosynthesis was elucidated. Very similar analogues of these compounds have been previously developed as inhibitors of juvenile hormone epoxide hydrolase (JHEH), a key enzyme in insect development and growth. Phurealipids also inhibit JHEH, and therefore phurealipids might contribute to bacterial virulence.

A comprehensive insight into the lipid composition of Myxococcus xanthus by UPLC-ESI-MS.

Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.

Characterisation of taxlllaids A-G; natural products from Xenorhabdus indica.

Six new lipodepsipeptides and an additional linear derivative named taxlllaids A-G (1-7) have been identified in the entomopathogenic bacterium Xenorhabdus indica. The structures of the main compounds have been solved by detailed NMR spectroscopic analysis and the structures of minor derivatives were elucidated by a combination of labelling experiments and detailed MS experiments. The absolute configuration of the taxlllaids was deduced by using the advanced Marfey method and analysis of the biosynthesis gene cluster showing the presence of epimerisation domains, which was subsequently proved to be correct by solid-phase peptide synthesis of all taxlllaids. The exchange of a single amino acid in the adenylation domain was shown to be responsible for substrate promiscuity of the third A domain, resulting in the incorporation of leucine, phenylalanine or tyrosine. Bioactivity testing revealed the taxlllaids to be weakly active against Plasmodium falciparum and against a number of eukaryotic cell lines.

Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

Bifurcation drives the evolution of assembly-line biosynthesis.

Reprogramming biosynthetic assembly-lines is a topic of intense interest. This is unsurprising as the scaffolds of most antibiotics in current clinical use are produced by such pathways. The modular nature of assembly-lines provides a direct relationship between the sequence of enzymatic domains and the chemical structure of the product, but rational reprogramming efforts have been met with limited success. To gain greater insight into the design process, we wanted to examine how Nature creates assembly-lines and searched for biosynthetic pathways that might represent evolutionary transitions. By examining the biosynthesis of the anti-tubercular wollamides, we uncover how whole gene duplication and neofunctionalization can result in pathway bifurcation. We show that, in the case of the wollamide biosynthesis, neofunctionalization is initiated by intragenomic recombination. This pathway bifurcation leads to redundancy, providing the genetic robustness required to enable large structural changes during the evolution of antibiotic structures. Should the new product be non-functional, gene loss can restore the original genotype. However, if the new product confers an advantage, depreciation and eventual loss of the original gene creates a new linear pathway. This provides the blind watchmaker equivalent to the design, build, test cycle of synthetic biology.

Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus.

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid's parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

Modification and de novo design of non-ribosomal peptide synthetases using specific assembly points within condensation domains.

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l-1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.

De novo design and engineering of non-ribosomal peptide synthetases.

Peptides derived from non-ribosomal peptide synthetases (NRPSs) represent an important class of pharmaceutically relevant drugs. Methods to generate novel non-ribosomal peptides or to modify peptide natural products in an easy and predictable way are therefore of great interest. However, although the overall modular structure of NRPSs suggests the possibility of adjusting domain specificity and selectivity, only a few examples have been reported and these usually show a severe drop in production titre. Here we report a new strategy for the modification of NRPSs that uses defined exchange units (XUs) and not modules as functional units. XUs are fused at specific positions that connect the condensation and adenylation domains and respect the original specificity of the downstream module to enable the production of the desired peptides. We also present the use of internal condensation domains as an alternative to other peptide-chain-releasing domains for the production of cyclic peptides.