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Synthetic Biology underpins advances in the bioeconomy

Biological systems - including the simplest cells - exhibit a broad range of functions to thrive in their environment. Research in the Imperial College Centre for Synthetic Biology is focused on the possibility of engineering the underlying biochemical processes to solve many of the challenges facing society, from healthcare to sustainable energy. In particular, we model, analyse, design and build biological and biochemical systems in living cells and/or in cell extracts, both exploring and enhancing the engineering potential of biology. 

As part of our research we develop novel methods to accelerate the celebrated Design-Build-Test-Learn synthetic biology cycle. As such research in the Centre for Synthetic Biology highly multi- and interdisciplinary covering computational modelling and machine learning approaches; automated platform development and genetic circuit engineering ; multi-cellular and multi-organismal interactions, including gene drive and genome engineering; metabolic engineering; in vitro/cell-free synthetic biology; engineered phages and directed evolution; and biomimetics, biomaterials and biological engineering.

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  • Journal article
    Cox R, Madsen C, McLaughlin J, Nguyen T, Roehner N, Bartley B, Bhatia S, Bissell M, Clancy K, Gorochowski T, Grunberg R, Luna A, Le Novere N, Pocock M, Sauro H, Sexton J, Stan G, Tabor J, Voigt C, Zundel Z, Myers C, Beal J, Wipat Aet al., 2018,

    Synthetic Biology Open Language Visual (SBOL Visual) Version 2.0

    , Journal of Integrative Bioinformatics, Vol: 15, ISSN: 1613-4516

    People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.0 of SBOL Visual, which builds on the prior SBOL Visual 1.0 standard by expanding diagram syntax to include functional interactions and molecular species, making the relationship between diagrams and the SBOL data model explicit, supporting families of symbol variants, clarifying a number of requirements and best practices, and significantly expanding the collection of diagram glyphs.

  • Journal article
    Elani Y, Trantidou T, Wylie D, Dekker L, Polizzi K, Law R, Ces Oet al., 2018,

    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules

    , Scientific Reports, Vol: 8, ISSN: 2045-2322

    There is increasing interest in constructing artificial cells by functionalisinglipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limitedin comparison to their biologicalcounterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger syntheticcell assembly. Using microfluidic technologies to construct such hybrid systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, whilethe cellacts as a bioreactor module that processes encapsulated feedstock which is further processedby a synthetic enzymatic cascadeco-encapsulated in the vesicle.

  • Journal article
    Jonas FRH, Royle KE, Aw R, Stan G, Polizzi KMet al., 2018,

    Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling

    , Synthetic and Systems Biotechnology, Vol: 3, Pages: 64-75, ISSN: 2405-805X

    Adaptation allows organisms to maintain a constant internal environment, which is optimised for growth. The unfolded protein response (UPR) is an example of a feedback loop that maintains endoplasmic reticulum (ER) homeostasis, and is characteristic of how adaptation is often mediated by transcriptional networks. The more recent discovery of asymmetric division in maintaining ER homeostasis, however, is an example of how alternative non-transcriptional pathways can exist, but are overlooked by gold standard transcriptomic or proteomic population-based assays. In this study, we have used a combination of fluorescent reporters, flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER homeostasis. Under low ER stress, asymmetric division leaves daughter cells with an ER deficiency, necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before growth. Mathematical analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter cells. These results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.

  • Journal article
    Broedel AK, Isalan M, 2018,

    Trp-ing upon new repressors

    , Nature Chemical Biology, Vol: 14, Pages: 328-329, ISSN: 1552-4450

    Bioengineers have used directed evolution to generate a new family of synthetic transcription factors based on the tryptophan repressor. The evolved repressor family enables researchers to build new gene circuits for biomedical applications.

  • Journal article
    Heide C, Ces O, Polizzi K, Kontoravdi Cet al.,

    Creating cell-free protein synthesis factories

    , Pharmaceutical Bioprocessing, ISSN: 2048-9145
  • Journal article
    Heide C, Ces O, Polizzi K, Kontoravdi Cet al.,

    Creating cell-free protein synthesis factories

    , Pharmaceutical Bioprocessing, ISSN: 2048-9145
  • Journal article
    Pothoulakis G, Ellis T, 2018,

    Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype

    , Communications Biology, Vol: 1, ISSN: 2399-3642

    Pseudohyphal growth is a multicellular phenotype naturally performed by wild budding yeast cells in response to stress. Unicellular yeast cells undergo gross changes in their gene regulation and elongate to form branched filament structures consisting of connected cells. Here, we construct synthetic gene regulation systems to enable external induction of pseudohyphal growth in Saccharomyces cerevisiae. By controlling the expression of the natural PHD1 and FLO8 genes we are able to trigger pseudohyphal growth in both diploid and haploid yeast, even in different types of rich media. Using this system, we also investigate how members of the BUD gene family control filamentation in haploid cells. Finally, we employ a synthetic genetic timer network to control pseudohyphal growth and further explore the reversibility of differentiation. Our work demonstrates that synthetic regulation can exert control over a complex multigene phenotype and offers opportunities for rationally modifying the resulting multicellular structure.

  • Journal article
    Niehus X, Crutz-LeCoq A-M, Sandoval G, Nicaud J-M, Ledesma Amaro Ret al., 2018,

    Engineering Yarrowia lipolytica to enhance lipid production from lignocellulosic materials

    , Biotechnology for Biofuels, Vol: 11, ISSN: 1754-6834

    Background: Yarrowia lipolytica is a common biotechnological chassis for the production of lipids, which are the pre‑ferred feedstock for the production of fuels and chemicals. To reduce the cost of microbial lipid production, inexpen‑sive carbon sources must be used, such as lignocellulosic hydrolysates. Unfortunately, lignocellulosic materials oftencontain toxic compounds and a large amount of xylose, which cannot be used by Y. lipolytica.Results: In this work, we engineered this yeast to efciently use xylose as a carbon source for the productionof lipids by overexpressing native genes. We further increased the lipid content by overexpressing heterologousgenes to facilitate the conversion of xylose-derived metabolites into lipid precursors. Finally, we showed that theseengineered strains were able to grow and produce lipids in a very high yield (lipid content = 67%, titer = 16.5 g/L,yield = 3.44 g/g sugars, productivity 1.85 g/L/h) on a xylose-rich agave bagasse hydrolysate in spite of toxiccompounds.Conclusions: This work demonstrates the potential of metabolic engineering to reduce the costs of lipid productionfrom inexpensive substrates as source of fuels and chemicals.

  • Journal article
    Mordaka PM, Heap JT, 2018,

    Stringency of synthetic promoter sequences in Clostridium revealed and circumvented by tuning promoter library mutation rates

    , ACS Synthetic Biology, Vol: 7, Pages: 672-681, ISSN: 2161-5063

    Collections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially relevant Clostridium spp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria like Clostridium. We directly compared oxygen-independent reporters of different types in Clostridium acetobutylicum and found that glucuronidase (GusA) from E. coli performed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus −35 and −10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthl was 25%. Surprisingly, none of the tested synthetic promoters from this library were functional in C. acetobutylicum, even though they functioned as expected in E. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthl was 79% or 58%, instead of 25% as before. Synthetic promoters from these “stringent” libraries functioned well in C. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly related species Clostridium sporogenes, and allowed predictable metabolic engineering of C. acetobutylicum for acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected “stringency” of promoter sequences in Clostridium, not reported previously.

  • Book chapter
    Lai H-E, Moore S, Polizzi K, Freemont Pet al., 2018,

    EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

    , Pages: 429-444

    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.

  • Journal article
    Khara DC, Schreck JS, Tomov TE, Berger Y, Ouldridge TE, Doye JPK, Nir Eet al., 2017,

    DNA bipedal motor walking dynamics: an experimental and theoretical study of the dependency on step size

    , Nucleic Acids Research, Vol: 46, Pages: 1553-1561, ISSN: 0305-1048

    We present a detailed coarse-grained computer simulation and single molecule fluorescence study of the walking dynamics and mechanism of a DNA bipedal motor striding on a DNA origami. In particular, we study the dependency of the walking efficiency and stepping kinetics on step size. The simulations accurately capture and explain three different experimental observations. These include a description of the maximum possible step size, a decrease in the walking efficiency over short distances and a dependency of the efficiency on the walking direction with respect to the origami track. The former two observations were not expected and are non-trivial. Based on this study, we suggest three design modifications to improve future DNA walkers. Our study demonstrates the ability of the oxDNA model to resolve the dynamics of complex DNA machines, and its usefulness as an engineering tool for the design of DNA machines that operate in the three spatial dimensions.

  • Journal article
    Broedel AK, Isalan M, Jaramillo A, 2017,

    Engineering of biomolecules by bacteriophage directed evolution

    , Current Opinion in Biotechnology, Vol: 51, Pages: 32-38, ISSN: 0958-1669

    Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein–protein interactions. Advances in this field expand the possible applications of protein and RNA engineering. This will ultimately result in new biomolecules with tailor-made properties, as well as giving us a better understanding of basic evolutionary processes.

  • Journal article
    Ouldridge TE, 2017,

    The importance of thermodynamics for molecular systems, and the importance of molecular systems for thermodynamics

    , Natural Computing, Vol: 17, Pages: 3-29, ISSN: 1567-7818

    Improved understanding of molecular systems has only emphasised thesophistication of networks within the cell. Simultaneously, the advance ofnucleic acid nanotechnology, a platform within which reactions can beexquisitely controlled, has made the development of artificial architecturesand devices possible. Vital to this progress has been a solid foundation in thethermodynamics of molecular systems. In this pedagogical review andperspective, I will discuss how thermodynamics determines both the overallpotential of molecular networks, and the minute details of design. I will thenargue that, in turn, the need to understand molecular systems is helping todrive the development of theories of thermodynamics at the microscopic scale.

  • Journal article
    Royle KE, Polizzi KM, 2017,

    A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris

    , Scientific Reports, Vol: 7, ISSN: 2045-2322

    Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.

  • Journal article
    Wintle BC, Boehm CR, Rhodes C, Molloy JC, Millett P, Adam L, Breitling R, Carlson R, Casagrande R, Dando M, Doubleday R, Drexler E, Edwards B, Ellis T, Evans NG, Hammond R, Haseloff J, Kahl L, Kuiken T, Lichman BR, Matthewman CA, Napier JA, Oheigeartaigh SS, Patron NJ, Perello E, Shapira P, Tait J, Takano E, Sutherland WJet al., 2017,

    A transatlantic perspective on 20 emerging issues in biological engineering

    , eLife, Vol: 6, ISSN: 2050-084X

    Advances in biological engineering are likely to have substantial impacts on global society. To explorethese potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunitiesand risks presented by biological engineering. We first identified 70 potential issues, and then used an iterativeprocess to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to berelatively unknown outside the field of biological engineering. The issues identified may be of interest toresearchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.

  • Journal article
    Park YK, Nicaud JM, Ledesma Amaro R, 2017,

    The engineering potential of Rhodosporidium toruloides as a workhorse for biotechnological applications

    , Trends in Biotechnology, Vol: 36, Pages: 304-317, ISSN: 0167-7799

    Moving our society towards a bioeconomy requires efficient and sustainable microbial production of chemicals and fuels. Rhodotorula (Rhodosporidium) toruloides is a yeast that naturally synthesizes substantial amounts of specialty chemicals and has been recently engineered to (i) enhance its natural production of lipids and carotenoids, and (ii) produce novel industrially relevant compounds. The use of R. toruloides by companies and research groups has exponentially increased in recent years as a result of recent improvements in genetic engineering techniques and the availability of multiomics information on its genome and metabolism. This review focuses on recent engineering approaches in R. toruloides for bioproduction and explores its potential as a biotechnological chassis.

  • Journal article
    Selles Vidal L, Kelly CL, Mordaka PM, Heap JTet al., 2017,

    Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application.

    , Biochimica et Biophysica Acta - Proteins and Proteomics, Vol: 1866, Pages: 327-347, ISSN: 1570-9639

    NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P)(+). NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilisation, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

  • Journal article
    Deshpande A, Ouldridge TE, 2017,

    High rates of fuel consumption are not required by insulating motifs to suppress retroactivity in biochemical circuits

    , Engineering Biology, Vol: 1, Pages: 86-99, ISSN: 2398-6182

    Retroactivity arises when the coupling of a molecular network $\mathcal{U}$to a downstream network $\mathcal{D}$ results in signal propagation back from$\mathcal{D}$ to $\mathcal{U}$. The phenomenon represents a breakdown inmodularity of biochemical circuits and hampers the rational design of complexfunctional networks. Considering simple models of signal-transductionarchitectures, we demonstrate the strong dependence of retroactivity on theproperties of the upstream system, and explore the cost and efficacy offuel-consuming insulating motifs that can mitigate retroactive effects. We findthat simple insulating motifs can suppress retroactivity at a low fuel cost bycoupling only weakly to the upstream system $\mathcal{U}$. However, this designapproach reduces the signalling network's robustness to perturbations from leakreactions, and potentially compromises its ability to respond torapidly-varying signals.

  • Journal article
    Larroude M, Celinska E, Back A, Thomas S, Nicaud JM, Ledesma Amaro Ret al., 2017,

    A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β-carotene

    , Biotechnology and Bioengineering, Vol: 115, Pages: 464-472, ISSN: 1097-0290

    The increasing market demands of β-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more β-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of β-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β-carotene.

  • Journal article
    Ogonah OW, Polizzi KM, Bracewell DG, 2017,

    Cell free protein synthesis: a viable option for stratified medicines manufacturing?

    , CURRENT OPINION IN CHEMICAL ENGINEERING, Vol: 18, Pages: 77-83, ISSN: 2211-3398

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