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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
    Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, Nolan T, Crisanti Aet al., 2018,

    A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

    , Nature Biotechnology, Vol: 36, Pages: 1062-1066, ISSN: 1087-0156

    In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

  • Journal article
    Girvan P, Teng X, Brooks NJ, Baldwin GS, Ying Let al., 2018,

    Redox Kinetics of the Amyloid-beta-Cu Complex and Its Biological Implications

    , BIOCHEMISTRY, Vol: 57, Pages: 6228-6233, ISSN: 0006-2960
  • Journal article
    Ledesma-Amaro R, Jiménez A, Revuelta JL, 2018,

    Pathway Grafting for Polyunsaturated Fatty Acids Production in Ashbya gossypii through Golden Gate Rapid Assembly.

    , ACS Synthetic Biology, Vol: 7, Pages: 2340-2347, ISSN: 2161-5063

    Here we present a Golden Gate assembly system adapted for the rapid genomic engineering of the industrial fungus Ashbya gossypii. This biocatalyst is an excellent biotechnological chassis for synthetic biology applications and is currently used for the industrial production of riboflavin. Other bioprocesses such as the production of folic acid, nucleosides, amino acids and biolipids have been recently reported in A. gossypii. In this work, an efficient assembly system for the expression of heterologous complex pathways has been designed. The expression platform comprises interchangeable DNA modules, which provides flexibility for the use of different loci for integration, selection markers and regulatory sequences. The functionality of the system has been applied to engineer strains able to synthesize polyunsaturated fatty acids (up to 35% of total fatty acids). The production of the industrially relevant arachidonic, eicosapentanoic and docosahexanoic acids remarks the potential of A. gossypii to produce these functional lipids.

  • Journal article
    Hurault G, Schram M, Roekevisch E, Spuls P, Tanaka RJet al., 2018,

    Relationship and probabilistic stratification of EASI and oSCORAD severity scores for atopic dermatitis

    , British Journal of Dermatology, Vol: 179, Pages: 1003-1005, ISSN: 1365-2133

    The Harmonizing Outcome Measures for Eczema (HOME) recommended the Eczema Area and Severity Index (EASI) as the core outcome instrument for measuring the clinical signs of atopic dermatitis (AD). However, EASI may not have been used in previous clinical trials, and other scores, e.g. SCORAD (SCORing Atopic Dermatitis), the objective component of SCORAD (oSCORAD) and the Investigator Global Assessment (IGA), remain widely used. It is useful to establish a method to convert these scores into EASI to compare the results from different studies effectively. Indeed, EASI and oSCORAD have been found to be strongly correlated (rSpearman=0.92)7, suggesting a possibility to find a relationship between the two scores.

  • Journal article
    Kelly CL, Harris AWK, Steel H, Hancock EJ, Heap JT, Papachristodoulou Aet al., 2018,

    Synthetic negative feedback circuits using engineered small RNAs

    , Nucleic Acids Research, Vol: 46, Pages: 9875-9889, ISSN: 0305-1048

    Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

  • Journal article
    Gorochowski TE, Ellis T, 2018,

    Designing efficient translation

    , NATURE BIOTECHNOLOGY, Vol: 36, Pages: 934-935, ISSN: 1087-0156
  • Journal article
    Trantidou T, Dekker L, Polizzi K, Ces O, Elani Yet al., 2018,

    Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

    , Interface Focus, Vol: 8, ISSN: 2042-8901

    The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.

  • Journal article
    Revuelta JL, Serrano-Amatriain C, Ledesma-Amaro R, Jiménez Aet al., 2018,

    Formation of folates by microorganisms: towards the biotechnological production of this vitamin

    , Applied Microbiology and Biotechnology, Vol: 102, Pages: 8613-8620, ISSN: 0175-7598

    Folates (vitamin B9) are essential micronutrients which function as cofactors in one-carbon transfer reactions involved in the synthesis of nucleotides and amino acids. Folate deficiency is associated with important diseases such as cancer, anemia, cardiovascular diseases, or neural tube defects. Epidemiological data show that folate deficiency is still highly prevalent in many populations. Hence, food fortification with synthetic folic acid (i.e., folic acid supplementation) has become mandatory in many developed countries. However, folate biofortification of staple crops and dairy products as well as folate bioproduction using metabolically engineered microorganisms are promising alternatives to folic acid supplementation. Here, we review the current strategies aimed at overproducing folates in microorganisms, in view to implement an economic feasible process for the biotechnological production of the vitamin.

  • Journal article
    Liu J, Li J, Liu Y, Shin H-D, Ledesma-Amaro R, Du G, Chen J, Liu Let al., 2018,

    Synergistic rewiring of carbon metabolism and redox metabolism in cytoplasm and mitochondria of aspergillus oryzae for increased l-Malate production

    , ACS Synthetic Biology, Vol: 7, Pages: 2139-2147, ISSN: 2161-5063

    l-Malate is an important platform chemical that has extensive applications in the food, feed, and wine industries. Here, we synergistically engineered the carbon metabolism and redox metabolism in the cytosol and mitochondria of a previously engineered Aspergillus oryzae to further improve the l-malate titer and decrease the byproduct succinate concentration. First, the accumulation of the intermediate pyruvate was eliminated by overexpressing a pyruvate carboxylase from Rhizopus oryzae in the cytosol and mitochondria of A. oryzae, and consequently, the l-malate titer increased 7.5%. Then, malate synthesis via glyoxylate bypass in the mitochondria was enhanced, and citrate synthase in the oxidative TCA cycle was downregulated by RNAi, enhancing the l-malate titer by 10.7%. Next, the exchange of byproducts (succinate and fumarate) between the cytosol and mitochondria was regulated by the expression of a dicarboxylate carrier Sfc1p from Saccharomyces cerevisiae in the mitochondria, which increased l-malate titer 3.5% and decreased succinate concentration 36.8%. Finally, an NADH oxidase from Lactococcus lactis was overexpressed to decrease the NADH/NAD+ ratio, and the engineered A. oryzae strain produced 117.2 g/L l-malate and 3.8 g/L succinate, with an l-malate yield of 0.9 g/g corn starch and a productivity of 1.17 g/L/h. Our results showed that synergistic engineering of the carbon and redox metabolisms in the cytosol and mitochondria of A. oryzae effectively increased the l-malate titer, while simultaneously decreasing the concentration of the byproduct succinate. The strategies used in our work may be useful for the metabolic engineering of fungi to produce other industrially important chemicals.

  • Journal article
    McFarlane C, Shah N, Kabasakal B, Cotton CAR, Bubeck D, Murray Jet al., 2018,

    Structural basis of light-induced redox regulation in the Calvin cycle

    , biorxiv

    Abstract In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. One Sentence Summary How plants turn off carbon fixation in the dark.

  • Journal article
    Trantidou T, Friddin M, Salehi-Reyhani S, Ces O, Elani Yet al., 2018,

    Droplet microfluidics for the construction of compartmentalised model membranes

    , Lab on a Chip, Vol: 18, Pages: 2488-2509, ISSN: 1473-0189

    The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.

  • Journal article
    Waters AJ, Capriotti P, Gaboriau DCA, Papathanos PA, Windbichler Net al., 2018,

    Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

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

    The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

  • Journal article
    Aw R, McKay P, Shattock R, Polizzi Ket al., 2018,

    A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization

    , Protein Expression and Purification, Vol: 149, Pages: 43-50, ISSN: 1046-5928

    Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L-1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.

  • Conference paper
    Puch A, Witkowski G, Isalan M, Mielcarek M, Zielonka Det al., 2018,

    A frequency of concomitant disorders in presymptomatic huntington’s disease patients

    , EHDN Plenary Meeting 2018, Publisher: BMJ Publishing Group, Pages: A42-A43, ISSN: 1468-330X
  • Conference paper
    Toczek M, Zielonka D, Marcinkowski J, Isalan M, Smolenski R, Mielcarek Met al., 2018,

    An altered metabolism of nucleotides leads to huntington’s disease related cardiomyopathy

    , EHDN Plenary Meeting, Publisher: BMJ Publishing Group, Pages: A13-A13, ISSN: 1468-330X
  • Journal article
    Yunus IS, Wichmann J, Wördenweber R, Lauersen KJ, Kruse O, Jones PRet al., 2018,

    Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel

    , Metabolic Engineering, Vol: 49, Pages: 201-211, ISSN: 1096-7176

    Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synt

  • Journal article
    O'Clery N, Yuan Y, Stan G-B, Barahona Met al., 2018,

    Global Network Prediction from Local Node Dynamics

    The study of dynamical systems on networks, describing complex interactiveprocesses, provides insight into how network structure affects globalbehaviour. Yet many methods for network dynamics fail to cope with large orpartially-known networks, a ubiquitous situation in real-world applications.Here we propose a localised method, applicable to a broad class of dynamicalmodels on networks, whereby individual nodes monitor and store the evolution oftheir own state and use these values to approximate, via a simple computation,their own steady state solution. Hence the nodes predict their own final statewithout actually reaching it. Furthermore, the localised formulation enablesnodes to compute global network metrics without knowledge of the full networkstructure. The method can be used to compute global rankings in the networkfrom local information; to detect community detection from fast, localtransient dynamics; and to identify key nodes that compute global networkmetrics ahead of others. We illustrate some of the applications of thealgorithm by efficiently performing web-page ranking for a large internetnetwork and identifying the dynamic roles of inter-neurons in the C. Elegansneural network. The mathematical formulation is simple, widely applicable andeasily scalable to real-world datasets suggesting how local computation canprovide an approach to the study of large-scale network dynamics.

  • Journal article
    Wu Y, Chen T, Liu Y, Lv X, Li J, Du G, Ledesma Amaro R, Liu Let al., 2018,

    CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis

    , Metabolic Engineering, Vol: 49, Pages: 232-241, ISSN: 1096-7176

    Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.

  • Journal article
    Schaerli Y, Jiménez A, Duarte JM, Mihajlovic L, Renggli J, Isalan M, Sharpe J, Wagner Aet al., 2018,

    Synthetic circuits reveal how mechanisms of gene regulatory networks constrain evolution

    , Molecular Systems Biology, Vol: 14, ISSN: 1744-4292

    Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such biases are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most pertinent evidence is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in Escherichia coli that produce a gene expression stripe—a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

  • Journal article
    Yunus IS, Jones PR, 2018,

    Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols

    , Metabolic Engineering, Vol: 49, Pages: 59-68, ISSN: 1096-7176

    Cyanobacteria can directly channel atmospheric CO2 into a wide range of versatile carbon products such as fatty acids and fatty alcohols with applications including fuel, cosmetics, and health products. Works on alcohol production in cyanobacteria have so far focused on either long (C12-C18) or short (C2-C4) chain-length products. In the present work, we report the first synthetic pathway for 1-octanol (C8) biosynthesis in Synechocystis sp. PCC 6803, employing a carboxylic acid reductase and C8-preferring fatty acyl-ACP thioesterase. The first engineered strain produced 1-octanol but exhibited poor productivity and cellular health issues. We therefore proceeded to systematically optimize the strain and cultivation conditions in order to understand what the limiting factors were. The identification of optimal promoters and ribosomal binding sites, in combination with isopropyl myristate solvent overlay, resulted in a combined (C8-OH and C10-OH) titer of more than 100 mg/L (a 25-fold improvement relative to the first engineered strain) and a restoration of cellular health. Additionally, more than 905 mg/L 1-octanol was produced when the strain expressing sfp (phosphopantetheinyl transferase) and car (carboxylic acid reductase) was fed with octanoic acid. A combination of feeding experiments and protein quantification indicated that the supply of octanoic acid from the introduced thioesterase, and possibly also native fatty acid synthesis pathway, were the main bottlenecks of the pathway.

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