Dr Simon Moore

Article

• Lai, H., Canavan, C., Cameron, L., Moore, S., Danchenko, M., Kuiken, T., Sekeyová, Z. and Freemont, P. (2019). Synthetic Biology and the United Nations. Trends in Biotechnology [Online]. Available at: https://doi.org/10.1016/j.tibtech.2019.05.011.
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  Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).
• Moore, S., MacDonald, J., Wienecke, S., Ishwarbhai, A., Tsipa, A., Aw, R., Kylilis, N., Bell, D., McClymont, D., Jensen, K., Polizzi, K., Biedendieck, R. and Freemont, P. (2018). Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria. Proceedings of the National Academy of Sciences [Online]. Available at: https://doi.org/10.1073/pnas.1715806115.
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  Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.
• Moore, S., MacDonald, J. and Freemont, P. (2017). Cell-free synthetic biology for in vitro prototype engineering. Biochemical Society Transactions [Online] 45:785-791. Available at: http://dx.doi.org/10.1042/BST20170011.
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  Cell-free transcription-translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test-kits for onsite identification of viruses (Zika, Ebola), whilst gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells.
• Moore, S., Sowa, S., Schuchardt, C., Deery, E., Lawrence, A., Ramos, J., Billig, S., Birkemeyer, C., Chivers, P., Howard, M., Rigby, S., Layer, G. and Warren, M. (2017). Elucidation of the biosynthesis of the methane catalyst coenzyme F430. Nature [Online]:78-82. Available at: http://dx.doi.org/10.1038/nature21427.
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  Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(II)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen III, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA–CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.
• Moore, S., Lai, H., Needham, H., Polizzi, K. and Freemont, P. (2017). Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool. Biotechnology Journal [Online] 12. Available at: https://doi.org/10.1002/biot.201600678.
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  Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the TetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, we demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.
• Moore, S., Lai, H., Kelwick, R., Chee, S., Bell, D., Polizzi, K. and Freemont, P. (2016). EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. ACS Synthetic Biology [Online] 5:1059-1069. Available at: https://doi.org/10.1021/acssynbio.6b00031.
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  Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology.
• Kopniczky, M., Moore, S. and Freemont, P. (2015). Multilevel Regulation and Translational Switches in Synthetic Biology. IEEE Transactions on Biomedical Circuits and Systems [Online] 9:485-496. Available at: https://doi.org/10.1109/TBCAS.2015.2451707.
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  In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules.Thisisabouttochangeastherepertoireoffoundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and ribonucleic acid (RNA) responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications.
• Moore, S., Mayer, M., Biedendieck, R., Deery, E. and Warren, M. (2014). Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements. New Biotechnology [Online] 31:553-561. Available at: http://dx.doi.org/10.1016/j.nbt.2014.03.003.
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  Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B12 (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiXL) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiXL in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiXL. From this we were able to show that the entire cbi operon is transcriptionally regulated by a B12-riboswitch, with a switch-off point at approximately 5 nM cobalamin. To bypass the effects of the B12-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200 ?g L?1). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials.
• Moore, S., Lawrence, A., Biedendieck, R., Deery, E., Frank, S., Howard, M., Rigby, S. and Warren, M. (2013). Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proceedings of the National Academy of Sciences [Online] 110:14906-14911. Available at: http://dx.doi.org/10.1073/pnas.1308098110.
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  It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.
• Moore, S., Biedendieck, R., Lawrence, A., Deery, E., Howard, M., Rigby, S. and Warren, M. (2013). Characterization of the enzyme CbiH60 involved in anaerobic ring contraction of the cobalamin (vitamin B12) biosynthetic pathway. Journal of Biological Chemistry [Online] 288:297-305. Available at: http://dx.doi.org/10.1074/jbc.M112.422535.
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  The anaerobic pathway for the biosynthesis of cobalamin (vitamin B(12)) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH(60), that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH(60) was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH(60) demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.
• Moore, S. and Warren, M. (2012). The anaerobic biosynthesis of vitamin B12. Biochemical Society Transactions [Online] 40:581-586. Available at: http://dx.doi.org/10.1042/BST20120066.
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  Vitamin B12 (cobalamin) is a cobalt-containing modified tetrapyrrole that is an essential nutrient for higher animals. Its biosynthesis is restricted to certain bacteria and requires approximately 30 enzymatic steps for its complete de novo construction. Remarkably, two distinct biosynthetic pathways exist, which are termed the aerobic and anaerobic routes. The anaerobic pathway has yet to be fully characterized due to the inherent instability of its oxygen-sensitive intermediates. Bacillus megaterium, a bacterium previously used for the commercial production of cobalamin, has a complete anaerobic pathway and this organism is now being used to investigate the anaerobic B12 pathway through the application of recent advances in recombinant protein production. The present paper provides a summary of recent findings in the anaerobic pathway and future perspectives.

Book section

• Lai, H., Moore, S., Polizzi, K. and Freemont, P. (2018). EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. In: Braman, J. C. ed. Synthetic Biology: Methods and Protocols. Springer. Available at: https://doi.org/10.1007/978-1-4939-7795-6_25.
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  Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.