Category Archives: Publications

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

The generation of “unNatural” products: Synthetic biology meets synthetic chemistry

Rebecca J. M. Goss ; Sreejith Shankar ; Antoine Abou Fayad

Nat Prod Rep 2012, 29 870-889

Natural product analogue generation is important, providing tools for chemical biology, enabling structure activity relationship determination and insight into the way in which natural products interact with their target biomolecules. The generation of analogues is also often necessary in order to improve bioavailability and to fine tune compounds’ activity. This review provides an overview of the catalogue of approaches available for accessing series of analogues. Over the last few years there have been major advances in genome sequencing and the development of tools for biosynthetic pathway engineering; it is therefore becoming increasingly easy to combine molecular biology and synthetic organic chemistry in order to enable expeditious access to series of natural products. This review outlines the various ways of combining biology and chemistry that have been applied to analogue generation, drawing upon a series of examples to illustrate each approach.

 

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.

A Single Streptomyces Symbiont Makes Multiple Antifungals to Support the Fungus Farming Ant Acromyrmex octospinosus

Ryan F. Seipke ; Joerg Barke ; Charles Brearley ; Lionel Hill ; Douglas W. Yu ; Rebecca J. M. Goss ; Matthew I. Hutchings

PLos ONE 2011, 6 (8) e22028

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 Streptomycesstrain that produces candicidin, consistent with another report that attine ants useStreptomyces-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 ~30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibitEscovopsis. 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.

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.

Total Synthesis of Chloptosin: A Dimeric Cyclohexapeptide

Alexander J. Oelke ; Francesca Antonietti ; Leonardo Bertone ; Philippa B. Cranwell ; David J. France ; Rebecca J. M. Goss ; Tatjana Hofmann ; Stephan Knauer ; Steven J. Moss ; Paul C. Skelton ; Richard M. Turner ; Georg Wuitschik ; Steven V. Ley

Chemistry – A European Journal 2011, 17 (15) 4183-4194

Here we describe in full our investigations into the synthesis of the dimeric cyclohexapeptide chloptosin in 17 linear steps. Particularly, this work features an organocatalytic tandem process for the synthesis of the embedded piperazic acids, in which a differentially protected azodicarboxylate is used together with pyrrolidinyl tetrazole as the catalyst. The central biaryl bond is being formed by Stille coupling of two sterically demanding ortho-chloropyrroloindole fragments. The inherent flexibility of the synthetic strategy proved beneficial as the route could be adjusted smoothly during the progression of the synthesis programme.

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

The saponins – polar isoprenoids with important and diverse biological activities

Anne Osbourn ; Rebecca J. M. Goss ; Robert A. Field

Nat Prod Rep 2011, 28 1261-1268

Saponins are polar molecules that consist of a triterpene or steroid aglycone with one or more sugar chains. They are one of the most numerous and diverse groups of plant natural products. These molecules have important ecological and agronomic functions, contributing to pest and pathogen resistance and to food quality in crop plants. They also have a wide range of commercial applications in the food, cosmetics and pharmaceutical sectors. Although primarily found in plants, saponins are produced by certain other organisms, including starfish and sea cucumbers. The underexplored biodiversity of this class of natural products is likely to prove to be a vital resource for discovery of high-value compounds. This review will focus on the biological activity of some of the best-studied examples of saponins, on the relationship between structure and function, and on prospects for synthesis of ‘‘designer’’ saponins.

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.