Prof Jason Micklefield PhD

Professor of Chemical Biology

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Research interests

The Micklefield lab is engaged in Chemical Biology and Synthetic Biology research tackling diverse challenges at the Chemistry-Biology interface utilising techniques and knowledge from organic chemistry and enzymology through to molecular microbiology and genetics. The main research themes include: 1) Biosynthesis and biosynthetic pathway engineering; 2) Biocatalysis and enzyme mechanism; 3) Nucleic acid redesign and recognition (Riboswitches).

1 Biosynthesis and Biosynthetic Engineering

We are interested in the biosynthesis and biosynthetic engineering of nonribosomal peptides, polyketides and other natural products. Most recently we discovered a new biosynthetic pathway, including a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line and a novel carboxylase enzyme (MloH), that produces a structurally unique antibiotic malonomycin [1]. This study, provides the first example of a vitamin-K dependent carboxylase (VKDC) enzyme in secondary metabolism and the first evidence for the function of VKDC-like proteins in prokaryotes. An interdisciplinary approach combining genome sequencing, bioinformatics, CRISPR-Cas9 gene editing, in vitro enzyme assays, 18O labeling, synthetic and analytical chemistry was used to elucidate the biosynthesis of malonomycin. The study also showed that many VKDC-like enzymes are present in bacteria, opening the way for the discovery of new pathways and novel antibiotics that are urgently required to combat emerging drug-resistant pathogens [1]. We also helped characterise the NRPS-PKS assembly line that delivers the epoxyketone proteasome inhibitor TMC-86A, which could be used to generate new antitumor agents [2]. Our lab has also recently developed new synthetic biology approaches to deliver improved natural product variants. For example, genes encoding two NRPS and a promiscuous tryptophan synthase from different species, were assembled in a heterologous host to create a de novo pathway to “non‐natural” thaxtomin phytotoxins, with improved stability, that are of industrial interest as herbicides for crop protection [3]. 

           

Earlier work in our lab focused on elucidating the biosynthetic origins of the calcium dependant antibiotics (CDA) from Streptomyces coelicolor, which are lipopeptides similar to daptomycin [4,5,6]. We went on to develop a wide range of biosynthetic engineering approaches (combinatorial biosynthesis) which enabled us to generate many "non-natural" lipopeptide variants by altering the specificity of the biosynthetic enzymes [6,7,8]. We have also developed methods for engineering the biosynthesis of the lipopeptide antibiotic enduracidin, using a membrane associated polyprenyl phosphomannose-dependent glycosyltransferases from ramoplanin biosynthesis to deliver novel lipoglycopeptide antibiotics [9].

[1] B. J. C. Law, Y. Zhuo, D. Francis, M. Winn, Y. Zhang, M. Samborskyy, A. Murphy, L. Ren, P. F. Leadlay & J. Micklefield. Nature Catalysis 20181, 977–984; [2] D. Zabala; J. W. Cartwright; D. M. Roberts; B. J. C. Law; L. Song; M. Samborskyy; P. F. Leadlay; J. Micklefield; G. L. Challis. J. Am. Chem. Soc2016138, 4342–4345; [3] M. Winn, D. Francis & J. Micklefield. Angew. Chem. Int. Ed. 201857, 6830–6833; [4] C. Milne, A. Powell, J. Jim, M. Al Nakeeb C. P. Smith & J. Micklefield. J. Am. Chem. Soc. 2006, 128, 11250-11259; [5] C. Mahlert, F. Kopp, J. Thirlway, J. Micklefield & M. A. Marahiel. J. Am. Chem. Soc. 2007, 129, 12011-12018; [6] A. Powell, M. Borg B. Amir-Heidari, J. M. Neary, J. Thirlway, B. Wilkinson, C.P. Smith and J. Micklefield. J. Am. Chem. Soc. 2007, 129, 15182-15191; [7]  J. Thirlway, R. Lewis, L. Nunns, M. Al Nakeeb, M. Styles, A.-W. Struck, C. P. Smith & J. Micklefield. Angew. Chem. Int. Ed. 2012, 51, 7181–7184; [8] R. Lewis, L. Nunns, J. Thirlway, K. Carroll, C. P. Smith, J. Micklefield. Chem. Commun. 2011, 47, 1860-1862; [9]  M.-C. Wu, M. Q. Styles, B. J. C. Law, A. W. Struck, L. Nunns and J. Micklefield. Microbiology 2015, 161, 1338-1347

2 Biocatalysis: Enzyme structure, mechanism and synthetic applications.

Our lab is also interested in the discovery, structure and mechanism of enzymes, to enable the further development and engineering to enzymes with improve properties for synthetic applications. For example, we employed structure guided mutagenesis and directed evolution to broaden the substrate scope and switch the regioselectivities of various bacterial and fungal halogenase enzymes [10, 11, 12, 13]. Our lab also demonstrated how halogenases can be integrated with palladium-catalyzed cross coupling chemistry, in one-pot reactions, to affect the direct regioselective arylation or alkenylation of C-H positions in various aromatic scaffolds [14]; such transformations are inaccessible using stand-alone chemo- or bio-catalysis.