Biosynthetic studies on fluoroacetate and longianone

This thesis explores the biosynthesis of two secondary metabolites, fluoroacetate and longianone, and involves the synthesis and feeding of deuterated putative intermediates. The bacterium Streptomyces cattleya produces fluoroacetate and 4- fluorothreonine; the mechanism by which C-F bond formation occurs is unknown. The stereochemistry of the fluorination event was investigated by feeding [2,2,3,3-(^-2)H(_4)]-succinate and (2R)-[l-(^2)H(_2)]- and (2S)-[l-(^2)H(_2)]-glycerols. The chirality of the resultant [2-(^2)H]-fluoroacetate was determined by chiral liquid crystal (^2)H-NMR and the fluorination was demonstrated to proceed with retention of stereochemistry. Longianone is produced by the slow growing fungus Xylaria longiana. This simple bicyclic metabolite is an isomer of the notorious fungal toxin patulin. Putative deuterated intermediates were administered to the fungus and the longianone produced analysed by (^2)H-NMR. It was demonstrated that longianone is biosynthesised from 6-methylsalicylic acid in a pathway closely related to that found in patulin biosynthesis

Fully aqueous and air compatible cross-coupling of primary alkyl halides with aryl boronic species : possible and facile

Funding: Engineering and Physical Sciences Research Council - EP/L016419/1; Royal Society.Publisher PDFPeer reviewe

The impact of viral infection on the chemistries of the Earth’s most abundant photosynthesisers : metabolically talented aquatic cyanobacteria

Funding: The authors thank BBSRC BB/T017058/1 (YW) IBioIC, MASTS, Xanthella (SF) and the Royal Society (RJMG) for financial support.Cyanobacteria are the most abundant photosynthesizers on earth, and as such, they play a central role in marine metabolite generation, ocean nutrient cycling, and the control of planetary oxygen generation. Cyanobacteriophage infection exerts control on all of these critical processes of the planet, with the phage-ported homologs of genes linked to photosynthesis, catabolism, and secondary metabolism (marine metabolite generation). Here, we analyze the 153 fully sequenced cyanophages from the National Center for Biotechnology Information (NCBI) database and the 45 auxiliary metabolic genes (AMGs) that they deliver into their hosts. Most of these AMGs are homologs of those found within cyanobacteria and play a key role in cyanobacterial metabolism-encoding proteins involved in photosynthesis, central carbon metabolism, phosphate metabolism, methylation, and cellular regulation. A greater understanding of cyanobacteriophage infection will pave the way to a better understanding of carbon fixation and nutrient cycling, as well as provide new tools for synthetic biology and alternative approaches for the use of cyanobacteria in biotechnology and sustainable manufacturing.Publisher PDFPeer reviewe

A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for > 60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in similar to 30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.Publisher PDFPeer reviewe

One-pot access to L-5,6-dihalotryptophans and L-alknyltryptophans using tryptophan synthase

The authors thank the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013/ERC grant agreement no 614779, and the University of St Andrews for a studentship (to D. R. M. Smith).We report, for the first time, the use of tryptophan synthase in the generation of L- dihalotryptophans and L-alkynyltryptophans. These previously unpublished compounds will be useful tools in the generation of probes for chemical biology, in biosynthetic diversification and as convenient building blocks for synthesis.PostprintPeer reviewe

Phospholipid composition and kinetics in different endobronchial fractions from healthy volunteers

Tracheal secretions may be of value as a surrogate to assess bronchoalveolar lavage fluid surfactant molecular composition and metabolism in healthy people. Despite minor differences, the phospholipid molecular composition of induced sputum also showed similarities to that of bronchoalveolar lavage fluid. Detailed analysis of newly synthesized individual phosphatidylcholine species provided novel insights into mechanisms of surfactant synthesis and acyl remodelling. Lysophosphatidylcholine methyl-D9 incorporation patterns suggest that these species are secreted together with other surfactant phospholipids and are not generated in the air spaces by hydrolysis of secreted surfactant phosphatidylcholine. Application into patient populations may elucidate potential underlying pathophysiological mechanisms that lead to surfactant alterations in disease state

Phenylalanine meta‐hydroxylase:A single residue mediates mechanistic control of aromatic amino acid hydroxylation

This work was supported by a project grant from the Biotechnology and Biological Sciences Research Council (BBSRC) U. K to R. J. M. G. (BB/I022910/2), and by the European Research Council under the European Union’s Seventh Framework Programme (FP7-3013/ERC grant agreement no 614779 GenoChemetics).The rare non-proteinogenic amino acid, meta- L-tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta-hydroxylation is unknown. Herein, we report the analysis of 3-hydroxylase (Phe3H) from Streptomyces coeruleorbidus. Insight from kinetic analyses, of both the wild-type enzyme and key mutants, of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advances understanding of the process by which meta-hydroxylation is mediated, revealing T202 to play an important role. In contrast to the established mechanism of tyrosine biogenesis, which proceeds via NIH shift, our data support direct, enzyme catalysed deprotonation following electrophilic aromatic substitution. We demonstrate that T202 is responsible for this shift in mechanism, with mutation to alanine resulting in a switch to the NIH shift mechanism and loss of regiospecificity. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta-L-tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates.Publisher PDFPeer reviewe

Halogenases:a palette of emerging opportunities for synthetic biology–synthetic chemistry and C–H functionalisation

Authors thank ERC GenoChemetics (FP7/2007-2013/ERC consolidator grant GCGXC grant agreement no. 614779 RJMG) for funding, BBSRC Follow on Funding, Synthetic Biology Highlight award. RSE Enterprise Fellowship (SVS), China Scholarship Council and EPSRC CRITICAT EP/L016419/1 for studentship support (Y. Z. & S. M. respectively).The enzymatic generation of carbon–halogen bonds is a powerful strategy used by both nature and synthetic chemists to tune the bioactivity, bioavailability and reactivity of compounds, opening up the opportunity for selective C–H functionalisation. Genes encoding halogenase enzymes have recently been shown to transcend all kingdoms of life. These enzymes install halogen atoms into aromatic and less activated aliphatic substrates, achieving selectivities that are often challenging to accomplish using synthetic methodologies. Significant advances in both halogenase discovery and engineering have provided a toolbox of enzymes, enabling the ready use of these catalysts in biotransformations, synthetic biology, and in combination with chemical catalysis to enable late stage C–H functionalisation. With a focus on substrate scope, this review outlines the mechanisms employed by the major classes of halogenases, while in parallel, it highlights key advances in the utilisation of the combination of enzymatic halogenation and chemical catalysis for C–H activation and diversification.Publisher PDFPeer reviewe

A one-pot synthesis of symmetrical and unsymmetrical dipeptide ureas

We gratefully acknowledge financial support from the BBSRC (Grant no. BB/I022910/1 and the European Commission (Seventh Framework Programme, Collaborative project "BlueGenics", Grant no. 311848).We describe a flexible and high yielding synthesis of 1,3-disubstituted ureas, that allows for the construction of both symmetrical and unsymmetrical dipeptide ureas, including easy access to 13C labelled ureas, from amino acids and carbon dioxide at atmospheric pressure.PostprintPostprintPeer reviewe

Regulation of surfactant protein D in the rodent prostate

<p>Abstract</p> <p>Background</p> <p>Surfactant protein D (SP-D) is an innate immune protein that is present in mucosal lined surfaces throughout the human body, including the male reproductive tract. In the present study, we characterized the regulation of SP-D expression in the mouse and rat prostate.</p> <p>Methods</p> <p>Real time reverse transcriptase polymerase chain reaction (RT-PCR) and immunostaining were used to characterize SP-D mRNA and protein in the mouse male reproductive tract. In order to evaluate the effects of testosterone on SP-D gene expression, we measured SP-D mRNA levels via real time RT-PCR in prostates from sham-castrated mice and castrated mice. In addition, we used a rat prostatitis model in which Escherichia coli was injected into the prostate in vivo to determine if infection influences SP-D protein levels in the prostate.</p> <p>Results</p> <p>We found that SP-D mRNA and protein are present throughout the mouse male reproductive tract, including in the prostate. We determined that castration increases prostate SP-D mRNA levels (~7 fold) when compared to levels in sham-castrated animals. Finally, we demonstrated that infection in the prostate results in a significant increase in SP-D content 24 and 48 hours post-infection.</p> <p>Conclusion</p> <p>Our results suggest that infection and androgens regulate SP-D in the prostate.</p