The Identification and Heterologous Expression of the Biosynthetic Gene Cluster Encoding the Antibiotic and Anticancer Agent Marinomycin.

With the rise in antimicrobial resistance, there is an urgent need for new classes of antibiotic with which to treat infectious disease. Marinomycin, a polyene antibiotic from a marine microbe, has been shown capable of killing methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), as well as having promising activity against melanoma. An attractive solution to the photoprotection of this antibiotic has been demonstrated. Here, we report the identification and analysis of the marinomycin biosynthetic gene cluster (BGC), and the biosynthetic assembly of the macrolide. The marinomycin BGC presents a challenge in heterologous expression due to its large size and high GC content, rendering the cluster prone to rearrangement. We demonstrate the transformation of Streptomyces lividans using a construct containing the cluster, and the heterologous expression of the encoded biosynthetic machinery and production of marinomycin B.

Expanding flavone and flavonol production capabilities in Escherichia coli.

Flavones and flavonols are important classes of flavonoids with nutraceutical and pharmacological value, and their production by fermentation with recombinant microorganisms promises to be a scalable and economically favorable alternative to extraction from plant sources. Flavones and flavonols have been produced recombinantly in a number of microorganisms, with Saccharomyces cerevisiae typically being a preferred production host for these compounds due to higher yields and titers of precursor compounds, as well as generally improved ability to functionally express cytochrome P450 enzymes without requiring modification to improve their solubility. Recently, a rapid prototyping platform has been developed for high-value compounds in E. coli, and a number of gatekeeper (2S)-flavanones, from which flavones and flavonols can be derived, have been produced to high titers in E. coli using this platform. In this study, we extended these metabolic pathways using the previously reported platform to produce apigenin, chrysin, luteolin and kaempferol from the gatekeeper flavonoids naringenin, pinocembrin and eriodictyol by the expression of either type-I flavone synthases (FNS-I) or type-II flavone synthases (FNS-II) for flavone biosynthesis, and by the expression of flavanone 3-dioxygenases (F3H) and flavonol synthases (FLS) for the production of the flavonol kaempferol. In our best-performing strains, titers of apigenin and kaempferol reached 128 mg L-1 and 151 mg L-1 in 96-DeepWell plates in cultures supplemented with an additional 3 mM tyrosine, though titers for chrysin (6.8 mg L-1) from phenylalanine, and luteolin (5.0 mg L-1) from caffeic acid were considerably lower. In strains with upregulated tyrosine production, apigenin and kaempferol titers reached 80.2 mg L-1 and 42.4 mg L-1 respectively, without the further supplementation of tyrosine beyond the amount present in the rich medium. Notably, the highest apigenin, chrysin and luteolin titers were achieved with FNS-II enzymes, suggesting that cytochrome P450s can show competitive performance compared with non-cytochrome P450 enzymes in prokaryotes for the production of flavones.

Harnessing intercellular signals to engineer the soil microbiome.

Covering: Focus on 2015 to 2020Plant and soil microbiomes consist of diverse communities of organisms from across kingdoms and can profoundly affect plant growth and health. Natural product-based intercellular signals govern important interactions between microbiome members that ultimately regulate their beneficial or harmful impacts on the plant. Exploiting these evolved signalling circuits to engineer microbiomes towards beneficial interactions with crops is an attractive goal. There are few reports thus far of engineering the intercellular signalling of microbiomes, but this article argues that it represents a tremendous opportunity for advancing the field of microbiome engineering. This could be achieved through the selection of synergistic consortia in combination with genetic engineering of signal pathways to realise an optimised microbiome.

Zelkovamycins B-E, Cyclic Octapeptides Containing Rare Amino Acid Residues from an Endophytic Kitasatospora sp.

Four unusual cyclopeptides, zelkovamycins B-E (1-4), were isolated from an endophytic Kitasatospora sp. Zelkovamycin B was featured by an unprecedented 3-methyl-5-hydroxypyrrolidine-2,4-dione ring system linked to the cyclopeptide skeleton. Their structures and full configurations were established by spectroscopic analysis, Marfey's method, and NMR calculations. A plausible biosynthetic pathway for zelkovamycins was proposed based on gene cluster analysis. Zelkovamycin E displayed potent inhibitory activity against H1N1 influenza A virus.

A marine viral halogenase that iodinates diverse substrates.

Oceanic cyanobacteria are the most abundant oxygen-generating phototrophs on our planet and are therefore important to life. These organisms are infected by viruses called cyanophages, which have recently shown to encode metabolic genes that modulate host photosynthesis, phosphorus cycling and nucleotide metabolism. Herein we report the characterization of a wild-type flavin-dependent viral halogenase (VirX1) from a cyanophage. Notably, halogenases have been previously associated with secondary metabolism, tailoring natural products. Exploration of this viral halogenase reveals it capable of regioselective halogenation of a diverse range of substrates with a preference for forming aryl iodide species; this has potential implications for the metabolism of the infected host. Until recently, a flavin-dependent halogenase that is capable of iodination in vitro had not been reported. VirX1 is interesting from a biocatalytic perspective as it shows strikingly broad substrate flexibility and a clear preference for iodination, as illustrated by kinetic analysis. These factors together render it an attractive tool for synthesis.

Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586.

The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU + macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A. MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A.

Fine Tuning of Antibiotic Activity by a Tailoring Hydroxylase in a Trans-AT Polyketide Synthase Pathway.

The addition or removal of hydroxy groups modulates the activity of many pharmacologically active biomolecules. It can be integral to the basic biosynthetic factory or result from associated tailoring steps. For the anti-MRSA antibiotic mupirocin, removal of a C8-hydroxy group late in the biosynthetic pathway gives the active pseudomonic acid A. An extra hydroxylation, at C4, occurs in the related but more potent antibiotic thiomarinol A. We report here in vivo and in vitro studies that show that the putative non-haem-iron(II)/α-ketoglutaratedependent dioxygenase TmuB, from the thiomarinol cluster, 4-hydroxylates various pseudomonic acids whereas C8-OH, and other substituents around the tetrahydropyran ring, block enzyme action but not substrate binding. Molecular modelling suggested a basis for selectivity, but mutation studies had a limited ability to rationally modify TmuB substrate specificity. 4-Hydroxylation had opposite effects on the potency of mupirocin and thiomarinol. Thus, TmuB can be added to the toolbox of polyketide tailoring technologies for the in vivo generation of new antibiotics in the future.

Expression of different bacterial cytotoxins is controlled by two global transcription factors, CRP and Fis, that co-operate in a shared-recruitment mechanism.

Pet is a cytotoxic autotransporter protein secreted by the pathogenic enteroaggregative Escherichia coli strain 042. Expression of Pet is co-dependent on two global transcription regulators: CRP (cyclic AMP receptor protein) and Fis (factor for inversion stimulation). At the pet promoter CRP binds to a single site centred at position -40.5 upstream of the start site for transcription. Due to the suboptimal positioning of this site, CRP alone activates transcription poorly and requires Fis to bind upstream to promote full activation. Here, we show that CRP and Fis control the expression of other important autotransporter toxins, namely Sat from uropathogenic E. coli (UPEC) and SigA from Shigella sonnei, and that this regulation has been conserved in different pathogens. Furthermore, we investigate the mechanism of Fis-mediated co-activation, exploiting a series of semi-synthetic promoters, with similar architecture to the pet promoter. We show that, when bound at position -40.5, CRP recruits RNA polymerase inefficiently and that Fis compensates by aiding polymerase recruitment through a direct protein-protein interaction. We demonstrate that other suitably positioned upstream transcription factors, which directly recruit RNA polymerase, can also compensate for the inappropriate positioning of CRP. We propose that this is a simple 'shared-recruitment' mechanism, by which co-dependence of promoters on two transcription factors could evolve.