Jason Micklefield

Jason Micklefield, PhD

Prof

 https://orcid.org/0000-0001-8951-4873

Personal profile

Biography

Jason Micklefield is Professor of Chemical Biology within the School of Chemistry and the Manchester Institute of Biotechnology. He graduated from the University of Cambridge in 1993 with a PhD in Chemistry, working with Prof Sir Alan R. Battersby FRS to complete the first total synthesis of haem d1. He then moved to the University of Washington, USA, as a NATO postdoctoral fellow investigating various biosynthetic pathways and enzyme mechanisms with Prof Heinz G. Floss. In 1995 he began his independent research career as a Lecturer in Organic Chemistry at Birkbeck College, University of London before moving to Manchester in 1998.

The Micklefield Lab develop more sustainable bio-inspired ways to build molecules. Our lab has an eclectic philosophy and is highly interdisciplinary, engaged in Chemical and Synthetic Biology research tackling diverse challenges at the Chemistry-Biology interface. We exploit techniques and knowledge from organic chemistry and enzymology through to molecular microbiology and genetics to develop sustainable routes to target molecules for therapeutic and other applications. The main research themes include: 1) Biosynthesis and biosynthetic pathway engineering providing novel bioactive natural products particularly new antibiotics to combat antimicrobial resistance (AMR) and treat neglected diseases; 2) Biocatalysis & integrated catalysis – Enzyme discovery, characterisation & engineering for enzymatic synthesis. Merging chemo- and biocatalysis for telescoping more sustainable routes to pharmaceuticals and other valuable products; 3) Nucleic acids chemistry and biology, including developing new routes to nucleic acid therapeutics (NAT) and functional tools such as riboswitches and aptamers.

Selected Publications

Research interests

The Micklefield Lab develop more sustainable bio-inspired ways to build molecules. Our lab has an eclectic philosophy and is highly interdisciplinary, engaged in Chemical and Synthetic Biology research tackling diverse challenges at the Chemistry-Biology interface. We exploit techniques and knowledge from organic chemistry and enzymology through to molecular microbiology and genetics to develop sustainable routes to target molecules for therapeutic and other applications. The main research themes include: 1) Biosynthesis and biosynthetic pathway engineering providing novel bioactive natural products particularly new antibiotics to combat antimicrobial resistance (AMR) and treat neglected diseases; 2) Biocatalysis & integrated catalysis - Enzyme discovery, characterisation & engineering for enzymatic synthesis. Merging chemo- and biocatalysis for telescoping more sustainable routes to pharmaceuticals and other valuable products; 3) Nucleic acids chemistry and biology, including developing new routes to nucleic acid therapeutics (NAT) and functional tools such as riboswitches and aptamers.

 

1) Biosynthesis and pathway engineering.

 

We are interested in the discovery, characterisation  and engineering of biosynthetic pathways to nonribosomal peptides, polyketides and other bioactive natural products. More  sustainable routes to the next generation of antibiotics to combat antimicrobial resistance (AMR) and treated neglected disease is the main focus. In addition, we have been working on pathways that produce natural products for agrochemical applications, which can help boost crop yields to feed the growing population. For example, we have characterised key ligase enzymes (CfaL) in the biosynthesis of coronatine and related bacterial phytotoxins that mimic the hormone jasmonyl-l-isoleucine ( JA-Ile), which mediates physiologically important plants [1]. Coronatine-like phytotoxins disrupt these essential pathways have potential in the development of safer, more selective herbicides.

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 [2]. 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 [2]. 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 [3].

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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 also of industrial interest as herbicides for crop protection [4].


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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 [5,6,7]. 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 [7,8,9]. 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 [10].

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[1] M. Winn, F. Wang, M. Rowlinson, L. Bering, D. Francis, C. Levy & J Micklefield Nature 2021, 593, 391–398; [2] 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 2018, 1, 977–984; [3] 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. Soc. 2016, 138, 4342–4345; [4] M. Winn, D. Francis & J. Micklefield. Angew. Chem. Int. Ed. 2018, 57, 6830–6833; [5] C. Milne, A. Powell, J. Jim, M. Al Nakeeb C. P. Smith & J. Micklefield. J. Am. Chem. Soc. 2006, 128, 11250-11259; [6] C. Mahlert, F. Kopp, J. Thirlway, J. Micklefield & M. A. Marahiel. J. Am. Chem. Soc. 2007, 129, 12011-12018; [7] 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; [8]  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; [9] R. Lewis, L. Nunns, J. Thirlway, K. Carroll, C. P. Smith, J. Micklefield. Chem. Commun. 2011, 47, 1860-1862; [10]  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 and integrated catalysis.

 

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.

Amide ligases: Most recently we discovered a new family of amide ligase enzymes (CfaL), which catalyse the synthesis of amides from a large variety of different carboxylic acid and amino acid substrates [1]. Amide bonds are fundamental in nature and are present in many of leading pharmaceuticals and other valuable products. Although chemical coupling of acids and amines is relatively simple, it often requires three steps, protect–couple–deprotect, to install each amide. Stoichiometric quantities of expensive and deleterious coupling reagents are typically required and purification can be problematic. We showed that CfaL have many advantages over traditional synthetic methods and other biocatalytic approaches, providing a cleaner, more sustainable, scalable and selective (obviate the need for protecting group), recyclable, tunable and less expensive route to important amides [1].

Solving the x-ray crystal structures CfaL enzymes provided valuable information which led to mutants, via structure guided mutagenesis, with improved stability and activities. We also showed how these ligases can be used for kinetic resolutions of racemic acid donor and amine acceptor substrates. Furthermore, we were able to demonstrate that these enzymes function efficiently at gram scale and preparation of precursors for important pharmaceuticals such as cancer treatments or potential COVID-19 drugs.

Halogenases & integrated catalysis: We have also been exploring the synthetic application of halogenase enzymes and integrated catalysis. 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 [2, 3, 4, 15]. 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 [6]; such transformations are inaccessible using stand-alone chemo- or bio-catalysis. We recently extended this concept, developing programmable, regioselective C−H bond functionalization methodologies for the installation of versatile nitrile, amide and carboxylic acid moieties through integration of halogenase enzymes with palladium-catalysed cyanation and subsequent incorporation of nitrile hydratase or nitrilase enzymes [7]. Using two- or three-component chemobiocatalytic systems, the regioselective synthesis of complex target molecules, including pharmaceuticals, can be achieved in a one-pot process operable on a gram scale.

Methyltransferase & alkyl diversification: S-adenosyl methionine (SAM)-dependent methyltransferase enzymes are also of major interest. We have  characterised and engineered various methyltransferases, to extend their substrate scope and to accept SAM analogues, with alternative substituents on the sulphonium centre,  demonstrating how these enzymes can be used in the alkyl-diversification of bioactive scaffolds [8, 9, 10, 11, 12]. We determined the effects of active site modification and quaternary structure on the regioselectivity of catechol-O-methyltransferase (COMT), as well as developing improved COMT enzymes for the production of vanillin and ethyl vanillin [8]. An enzyme cascade including COMT and tyrosinase (an hydroxylase) was also developed that enables the selective derivatisation of tyrosine residues in peptides and proteins using SAM analogues for potential labelling, imaging and therapeutic applications [9].

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Our lab also characterised the methyltransferase RapM demonstrating how it can be used for the  regioselective alkylation of the important immunosuppressive agent rapamycin [10]. In addition, we obtained the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture [11]. CNMT was then used in the alkyl-diversification of privileged tetrahydroisoquinoline pharmacophores [12].

We also showed how MT enzymes can utilise a naturally occurring SAM analogues carboxy-SAM (cxSAM) which is involved in carboxylmethylation of a tRNA substrate. We used cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates using engineered CNMT and COMT enzymes. Site-directed mutagenesis was used to create orthogonal CNMT and COMT variants possessing improved catalytic activity and selectivity for cxSAM. The subsequent coupling of these orthogonal MT, with the cxSAM generating enzyme CmoA, resulted in more efficient and selective carboxymethylation pathways. An enzymatic approach was also developed to generate a previously undescribed co-factor, carboxy-S-adenosyl-l-ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1-carboxyethyl group to the substrate.

Other examples of enzymatic synthesis: Our team demonstrated how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of L‐threonine and various indoles to generate β‐methyltryptophans and derivatives in a single step [13]. In earlier studies, we elucidated the structure, mechanism and developed directed evolution approaches to extend the biocatalytic repertoire of the aryl malonate decarboxylases [14, 15] Finally we have provided the first example of a temperature dependent switch in enzyme class, showing how phenylalanine aminomutase enzymes become lyase enzymes at elevated temperatures [16]

[1] M. Winn, F. Wang, M. Rowlinson, L. Bering, D. Francis, C. Levy & J Micklefield Nature 2021, 593, 391–398; [2] J. Latham, E. Brandenburger, S. A. Shepherd, B.R.K. Menon & J.Micklefield. Chem. Rev. 2017, 118, 232-269; [3] R. K. Menon, E. Brandenburger, H. H. Sharif, U. Klemstein, S.A. Shepherd, M. F. Greaney & J. Micklefield. Angew. Chem. Int. Ed. 2017, 56, 11841 –11845; [4] S. A. Shepherd, C. Karthikeyan, J. Latham, A.-W. Struck, M. L. Thompson, B. Menon, M. Styles, C. Levy, D. Leys & J. Micklefield. Chemical Science2015, 6, 3454-3460; [5] S. A. Shepherd, B. R. K. Menon, H. Fisk, A.-W. Struck, C. Levy, D. Leys & J. Micklefield. ChemBioChem2016, 17, 821–824; [6] J. Latham, J.-M. Henry, H. H. Sharif, B. R. K. Menon, S. A. Shepherd,  M. F. Greaney & J.Micklefield. Nature Commun.2016, 7, 11873; [7] E. J. Craven, J. Latham, S. A. Shepherd, I. Khan, A. Diaz-Rodriguez, M. F. Greaney & J. Micklefield Nature Catalysis 2021, 4, 385–394; [8] M. L. Thompson, C. Levy, S. A. Shepherd, D. Leys, and J. Micklefield Angew. Chem. Int. Ed. 2016, 55, 2683-2687; [9] A.-W. Struck, M. R. Bennett, S. A. Shepherd, B. J. C. Law, Y. Zhuo, L. S. Wong, J. Micklefield. J. Am. Chem. Soc.2016, 138, 3038-3045; [10] B. J. C. Law, A.-W. Struck, M. R. Bennett, B. Wilkinson and J. Micklefield. Chemical Science 2015, 6, 2885-2892; [11] M. R. Bennett, M. L. Thompson, S. A. Shepherd, M. S. Dunstan, A. J. Herbert, D. R. M. Smith, V. A. Cronin, B. R. K. Menon, C. Levy & J. Micklefield Angew. Chem. Int. Ed.2018, 57, 10600-10604; [12] A.  J. Herbert, S. A. Shepherd, V. A. Cronin, M. R. Bennett, R. Sung & J Micklefield Angew. Chem. Int. Ed. 2020, 59, 2–9; [13] D. Francis, M. Winn, J. Latham, M. F. Greaney & J. Micklefield. ChemBioChem.2017, 18, 382-386; [14] K. Okrasa, C. Levy, M. Wilding, M. Goodall, N. Baudendistel. B. Hauer, D. Leys, J. Micklefield. Angew. Chem. Int. Ed.2009, 48, 7691-7694; [15] R. Lewin, M. Goodall, M. L.Thompson, J. Leigh, M. Breuer, K. Baldenius & J. Micklefield. Chem. Eur. J.,2015, 17, 6557-6563; [16] C. Chesters, M. Wilding, M. Goodall, J. Micklefield.  Angew. Chem. Int. Ed. 2012, 51, 4344-4348.

 

3) Nucleic acid chemistry & biology.

 

Our lab has worked on nucleic acid chemistry and biology for many years.  In fact the first work published by our lab [1, 2] focused on the chemical synthesis and evaluation of novel backbone modified nucleic acids as antisense agents and in other applications [e.g. 1, 2, 3, 4]. Currently we are developing new and more sustainable ways of producing new modified nucleic acids for range of different important therapeutics uses. Our lab also has many years of experience in the enzymatic synthesis of nucleic acids. Notably we developed the first orthogonal riboswitches and aptamers (RNA elements) that recognise small molecule ligands for in vivo applications as gene expression tools and as biosensors.

Riboswitches, present within mRNA, control gene expression in response to specific metabolites. We succeeded in re-engineering the first orthogonal riboswitches that are no longer triggered by the natural metabolites, but instead could be controlled by the addition of various synthetic heterocyclic molecules [5]. Our re-engineering approach provides a methodological platform for the creation of new orthogonal regulatory components and biosensors for synthetic biology and biotechnological applications including gene functional analysis, antimicrobial target validation and screening. For example, we have shown how mutually orthogonal synthetic riboswitches can be used to affect the simultaneous and independent control of multiple genes within the same cell [6]. In addition, we have demonstrated how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules [7]. Our novel riboswitch–ligand pairings are shown to regulate physiologically important genes required for bacterial motility in E. coli, as well as cell morphology in B. subtilis [7]. A rational targeted approach was also used to re-engineer the PreQ1 riboswitch from B. subtilis, into an orthogonal OFF-switch that can be controlled by the addition of a synthetic ligand [8].

[1] K. J. Fettes, N. Howard, D. T. Hickman, S. A. Adah, M. R. Player, P. F. Torrence and J. Micklefield. Chem. Commun. 2000, 765-766; [2] D. T. Hickman, P. M. King, M. A. Cooper, J. M. Slater and J. Micklefield. Chem. Commun. 2000, 2251-225; [3] R. Worthington, J. Micklefield Chem. Eur. J.2011, 17, 14429-14441; [4] N. Bell, R. Wong, J. Micklefield  Chem. Eur. J.  2010, 16, 2026-2030; [5] N. Dixon, J. Duncan, T. Geerlings, M. S. Dunstan, J. McCarthy, D. Leys & J. Micklefield. Proc. Natl. Acad. Sci. USA 2010, 107, 2830 [6] N Dixon, C Robinson, J. N. Duncan, T Geerlings, S. P. Drummond & J. Micklefield. Angew. Chem. Int. Ed. 2012, 51, 3620; [7] C. J. Robinson, H. A. Vincent, M.-C. Wu, P. T. Lowe, M. S. Dunstan, D. Leys, J. Micklefield. J. Am. Chem. Soc. 2014, 136, 10615; [8] M. C. Wu, P. T. Lowe, C. J. Robinson, H. A. Vincent, N. Dixon, J. Leigh & J. Micklefield. J. Am. Chem. Soc. 2015, 137, 901

 

 

 

 

 

Opportunities

1. PhD Positions Currently Available

BBSRC PhD Case Studentship in the Micklefield Lab in collaboration with Exactmer  – Enzymatic methods for assembly of nucleic acid therapeutic agents.

BBSRC PhD Studentship in the Micklefield Lab – Bioengineering sustainable routes to anti-infective agents to combat AMR & future pandemics.

2. Postgraduate Opportunities General Information

Whilst research in the group encompasses aspects of both Chemistry and Biology, it is not expected or indeed required that individuals are trained in both areas, before they join the group. Indeed individuals that have a strong background in either chemistry or biological science and not both are preferred. Typically we recruit from two pools:

Candidates who are strong in core chemistry (e.g. 1 or 2i MChem or BSc in Chemistry or related degree). This includes those interested in any aspect of organic (e.g. synthesis), biological or analytical chemistry. Candidates who can demonstrate a wide knowledge of chemistry and possess an open mind are particularly encouraged. 

Candidates with strong degrees (e.g. 1 or 2i ) in biological sciences of a more molecular nature. Individuals with interests or experience in biochemistry and/or molecular biology are particularly encouraged. In this case the candidate does not need a strong background in basic chemistry.
 

3. Group Members

The Micklefield lab is composed of members from different backgrounds including chemistry, biochemistry, molecular biology, genetics and bioinformatics, forming a multidisciplinary group with expertise in a wide variety of disciplines.

Full list of current and former group members

Micklefield Lab 2018

Micklefield Lab 2019

Micklefield Lab 2016

Micklefield Lab 2014

 

Jason Micklefield Group 2013

Micklefield Lab 2012

 

Expertise related to UN Sustainable Development Goals

In 2015, UN member states agreed to 17 global Sustainable Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all. This person’s work contributes towards the following SDG(s):

  • SDG 3 - Good Health and Well-being
  • SDG 4 - Quality Education
  • SDG 7 - Affordable and Clean Energy
  • SDG 9 - Industry, Innovation, and Infrastructure
  • SDG 12 - Responsible Consumption and Production
  • SDG 15 - Life on Land

External positions

Visiting Professor, East China University Of Science & Technology

1 Apr 2018 → Apr 2021

Research Beacons, Institutes and Platforms

  • Sustainable Futures
  • Christabel Pankhurst Institute
  • Manchester Institute of Biotechnology

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