Category Archives: Former Lab Members

Glycosyltransferases from Oat (Avena) Implicated in the Acylation of Avenacins

Amorn Owatworakit ; Belinda Townsend ; Thomas Louveau ; Helen Jenner ; Martin Rejzek ; Richard K. Hughes ; Gerhard Saalbach ; Xiaoquan Qi ; Saleha Bakht ; Abhijeet Deb Roy ; Sam T. Mugford ; Rebecca J. M. Goss ; Robert A. Field ; Anne Osbourn

J Biol Chem 2013, 288 (6) 3696-3704

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avenaspp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is anN-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.


Crystallization and preliminary X-ray analysis of Pac17 from the pacidamycin-biosynthetic cluster of Streptomyces coeruleorubidus

Daniel R. Tromans ; Clare E. M. Stevenson ; Rebecca J. M. Goss ; David M. Lawson

Acta Cryst 2012, F68 971-974

Pac17 is an uncharacterized protein from the pacidamycin gene cluster of the soil bacterium Streptomyces coeruleorubidus. It is implicated in the biosynthesis of the core diaminobutyric acid residue of the antibiotic, although its precise role is uncertain at present. Given that pacidamycins inhibit translocase I of Pseudomonas aeruginosa, a clinically unexploited antibiotic target, they offer new hope in the search for antibacterial agents directed against this important pathogen. Crystals of Pac17 were grown by vapour diffusion and X-ray data were collected at a synchrotron to a resolution of 1.9 Å from a single crystal. The crystal belonged to space group C2, with unit-cell parameters a = 214.12, b = 70.88, c = 142.22 Å, [beta] = 92.96°. Preliminary analysis of these data suggests that the asymmetric unit consists of one Pac17 homotetramer, with an estimated solvent content of 49.0%.


Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy

Andreas N. Tsoligkas ; James Bowen ; Michael Winn ; Rebecca J. M. Goss ; Tim W. Overton ; Mark J. H. Simmons

Colloids and Surfaces B: Biointerfaces 2012, 89 152-160

The ability of biofilms to withstand chemical and physical extremes gives them the potential to be developed as robust biocatalysts. Critical to this issue is their capacity to withstand the physical environment within a bioreactor; in order to assess this capability knowledge of their surface properties and adhesive strength is required. Novel atomic force microscopy experiments conducted under growth conditions (30 °C) were used to characterise Escherichia coli biofilms, which were generated by a recently developed spin-coating method onto a poly-l-lysine coated glass substrate. High-resolution topographical images were obtained throughout the course of biofilm development, quantifying the tip–cell interaction force during the 10 day maturation process. Strikingly, the adhesion force between the Si AFM tip and the biofilm surface increased from 0.8 nN to 40 nN within 3 days. This was most likely due to the production of extracellular polymer substance (EPS), over the maturation period, which was also observed by electron microscopy. At later stages of maturation, multiple retraction events were also identified corresponding to biofilm surface features thought to be EPS components. The spin coated biofilms were shown to have stronger surface adhesion than an equivalent conventionally grown biofilm on the same glass substrate.

Biofilms and their engineered counterparts: A new generation of immobilised biocatalysts

Michael Winn ; Joanne M. Foulkes ; Stefano Perni ; Mark J. H. Simmons ; Tim W. Overton ; Rebecca J. M. Goss

Cat Sci Tech 2012, 2 1544-1547

The robust nature of biofilms makes them medicinally difficult to treat, however this same property renders them an attractive method for protecting and immobilising enzymes for biotransformation. Although biofilms consisting of a consortium of different microbial species have been routinely used in water purification for many decades, there are few reported examples of single species biofilms being harnessed for industrial applications. The potential of using tailored single species biofilms in order to catalyse a biotransformation of choice is attractive; we reflect upon recent advances in the use and generation of such platforms, from both biological and process engineering viewpoints.

Graphical abstract: Biofilms and their engineered counterparts: A new generation of immobilised biocatalysts

Biogenesis of the Unique 4 ‘,5 ‘-Dehydronucleoside of the Uridyl Peptide Antibiotic Pacidamycin

Amany E. Ragab ; Sabine Grüschow ; Daniel R. Tromans ; Rebecca J. M. Goss

JACS 2011, 133 (39) 15288-15291

The pacidamycins belong to a class of antimicrobial nucleoside antibiotics that act by inhibiting the clinically unexploited target translocase I, a key enzyme in peptidoglycan assembly. As with other nucleoside antibiotics, the pacidamycin 4′,5′-dehydronucleoside portion is an essential pharmacophore. Here we show that the biosynthesis of the pacidamycin nucleoside in Streptomyces coeruleorubidus proceeds through three steps from uridine. The transformations involve oxidation of the 5′-alcohol by Pac11, transamination of the resulting aldehyde by Pac5, and dehydration by the Cupin-domain protein Pac13.

Engineering Biofilms for Biocatalysis

Andreas N. Tsoligkas ; Michael Winn ; James Bowen ; Tim W. Overton ; Mark J. H. Simmons ; Rebecca J. M. Goss

ChemBioChem 2011, 12 (9) 1391-1395

Biofilm, friend not foe: Single species biofilms can be engineered to form robust biocatalysts with greater catalytic activity and significantly improved catalytic longevity than purified and immobilised enzymes. We report the engineering, structural analysis and biocatalytic capability of a biofilm that can mediate the conversion of serine and haloindoles to halotryptophans.

Diversity in natural product families is governed by more than enzyme promiscuity alone : establishing control of the pacidamycin portfolio

Sabine Grüschow ; Emma J. Rackham ; Rebecca J. M. Goss

Chem Sci 2011, 2 2182-2186

As with many other antibiotics, pacidamycins are produced as a suite of related compounds. Unlike most other secondary metabolites, however, this diversity is not solely the result of the substrate promiscuity of the biosynthetic enzymes but also arises from a gene duplication event (Pac21, Pac21h) and control of the precursor pool (PhhA). We are demonstrating the ability to harness these three levels of control in order to direct the selective production of specific members of this family of metabolites in a “dial-a-molecule” fashion. Furthermore, PhhA is shown to be a phenylalanine 3-hydroxylase, the first of the iron- and tetrahydropterin-dependentaromatic amino acid hydroxylases to be characterised with this regioselectivity.

Graphical abstract: Diversity in natural product families is governed by more than enzyme promiscuity alone: establishing control of the pacidamycin portfolio

Gene Expression Enabling Synthetic Diversification of Natural Products : Chemogenetic Generation of Pacidamycin Analogs

Abhijeet Deb Roy ; Sabine Grueschow ; Nickiwe Cairns ; Rebecca J. M. Goss

JACS 2010, 132 (35) 12243-12245

Introduction of prnA, the halogenase gene from pyrrolnitrin biosynthesis, into Streptomyces coeruleorubidus resulted in efficient in situ chlorination of the uridyl peptide antibotic pacidamycin. The installed chlorine provided a selectably functionalizable handle enabling synthetic modification of the natural product using mild cross-coupling conditions in crude aqueous extracts of the culture broth.

Pacidamycin Biosynthesis : Identification and Heterologous Expression of the First Uridyl Peptide Antibiotic Gene Cluster

Emma J. Rackham ; Sabine Grüschow ; Amany E. Ragab ; Shilo Dickens ; Rebecca J. M. Goss

ChemBioChem 2010, 11 (12) 1700-1709

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3′-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.

An Expeditious Route to Fluorinated Rapamycin Analogues by Utilising Mutasynthesis

Rebecca J. M. Goss ; Simon Lanceron ; Abhijeet Deb Roy ; Simon Sprague ; Mohammed Nur-e-Alam ; David L. Hughes ; Barrie Wilkinson ; Steven J. Moss

ChemBioChem 2010, 11 (5) 698-702

Rapamycin is a drug with several important clinical uses. Its complex structure means that total synthesis of this natural product and its analogues is demanding and lengthy. A more expeditious approach is to utilise biosynthesis to enable the generation of otherwise synthetically intractable analogues. In order to achieve this, rules governing biosynthetic precursor substrate preference must be established. Through determining these rules and synthesising and administering suitable substrate precursors, we demonstrate the first generation of fluorinated rapamycin analogues. Here we report the generation of six new fluororapamycins.