Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

Bioinformatic and Mechanistic Analysis of the Palmerolide PKS-NRPS Biosynthetic Pathway From the Microbiome of an Antarctic Ascidian

Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the ∼75 kbp trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicihabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

Advances in Biosynthesis of Natural Products from Marine Microorganisms

Natural products play an important role in drug development, among which marine natural products are an underexplored resource. This review summarizes recent developments in marine natural product research, with an emphasis on compound discovery and production methods. Traditionally, novel compounds with useful biological activities have been identified through the chromatographic separation of crude extracts. New genome sequencing and bioinformatics technologies have enabled the identification of natural product biosynthetic gene clusters in marine microbes that are difficult to culture. Subsequently, heterologous expression and combinatorial biosynthesis have been used to produce natural products and their analogs. This review examines recent examples of such new strategies and technologies for the development of marine natural products.

Isolation and identification of marine microbial products

Background The ocean is one of the world’s most important sources of bioactive chemicals in the marine environment. Microbiologists, ecologists, agronomists, taxonomists, and evolutionary biologists have been increasingly interested in marine microbial natural products (MMNPs) in recent decades. Main body Diverse marine bacteria appear to get the ability to manufacture an astounding diversity of MMNPs with a wide range of biological actions, including anti-tumor, antimicrobial, and anti-cardiovascular agents according to numerous studies. Short conclusions Innovative isolation and culture methodologies, tactics for identifying novel MMNPs via routine screens, metagenomics, genomics, combinatorial biosynthesis, and synthetic biology are all discussed in this review. There is also a discussion of potential issues and future directions for studying MMNPs.

Genome mining of novel rubiginones from Streptomyces sp. CB02414 and characterization of the post-PKS modification steps in rubiginone biosynthesis

Background Rubiginones belong to the angucycline family of aromatic polyketides, and they have been shown to potentiate the vincristine (VCR)-induced cytotoxicity against VCR-resistant cancer cell lines. However, the biosynthetic gene clusters (BGCs) and biosynthetic pathways for rubiginones have not been reported yet. Results In this study, based on bioinformatics analysis of the genome of Streptomyces sp. CB02414, we predicted the functions of the two type II polyketide synthases (PKSs) BGCs. The rub gene cluster was predicted to encode metabolites of the angucycline family. Scale-up fermentation of the CB02414 wild-type strain led to the discovery of eight rubiginones, including five new ones (rubiginones J, K, L, M, and N). Rubiginone J was proposed to be the final product of the rub gene cluster, which features extensive oxidation on the A-ring of the angucycline skeleton. Based on the production profiles of the CB02414 wild-type and the mutant strains, we proposed a biosynthetic pathway for the rubiginones in CB02414. Conclusions A genome mining strategy enabled the efficient discovery of new rubiginones from Streptomyces sp. CB02414. Based on the isolated biosynthetic intermediates, a plausible biosynthetic pathway for the rubiginones was proposed. Our research lays the foundation for further studies on the mechanism of the cytochrome P450-catalyzed oxidation of angucyclines and for the generation of novel angucyclines using combinatorial biosynthesis strategies.

Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis

Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2–7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.

Discovery and Heterologous Production of New Cyclic Depsibosamycins

Streptomyces are producers of valuable secondary metabolites with unique scaffolds that perform a plethora of biological functions. Nonribosomal peptides are of special interest due to their variety and complexity. They are synthesized by nonribosomal peptide synthetases, large biosynthetic machineries that are encoded in the genome of many Streptomyces species. The identification of new peptides and the corresponding biosynthetic gene clusters is of major interest since knowledge can be used to facilitate combinatorial biosynthesis and chemical semisynthesis of natural products. The recently discovered bosamycins are linear octapeptides with an interesting 5-OMe tyrosine moiety and various modifications at the N-terminus. In this study, the new cyclic depsibosamycins B, C, and D from Streptomyces aurantiacus LU19075 were discovered. In comparison to the linear bosamycins B, C, and D, which were also produced by the strain, the cyclic depsibosamycins showed a side-chain-to-tail lactonization of serine and glycine, leading to a ring of four amino acids. In silico identification and heterologous expression of the depsibosamycin (dbm) gene cluster indicated that the cyclic peptides, rather than the linear derivatives, are the main products of the cluster.

Palmerolide PKS-NRPS gene clusters characterized from the microbiome of an Antarctic ascidian

Polyketides are a complex family of natural products that often serve competitive or pro-survival purposes but can also demonstrate bioactivity in human diseases as, for example cholesterol lowering agents, anti-infectives, or anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGC) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A putative palmerolide BGC was identified from a S. adareanum metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, and active site and motif identification were used to confirm the identity and propose the function of the 75 kb trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and multiple copies found within the metagenome-assembled genome (MAG) of Candidatus Synoicohabitans palmerolidicus. Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic species. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.