Research

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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
    Vivek A, Bolognesi G, Elani Y, 2020,

    Fusing artificial cell compartments and lipid domains using optical traps: a tool to modulate membrane composition and phase behaviour

    , Micromachines, Vol: 11, ISSN: 2072-666X

    New technologies for manipulating biomembranes have vast potential to aid the understanding of biological phenomena, and as tools to sculpt novel artificial cell architectures for synthetic biology. The manipulation and fusion of vesicles using optical traps is amongst the most promising due to the level of spatiotemporal control it affords. Herein, we conduct a suite of feasibility studies to show the potential of optical trapping technologies to (i) modulate the lipid composition of a vesicle by delivering new membrane material through fusion events and (ii) manipulate and controllably fuse coexisting membrane domains for the first time. We also outline some noteworthy morphologies and transitions that the vesicle undergoes during fusion, which gives us insight into the mechanisms at play. These results will guide future exploitation of laser-assisted membrane manipulation methods and feed into a technology roadmap for this emerging technology.
  • Journal article
    Ukegbu CV, Giorgalli M, Tapanelli S, Rona LDP, Jaye A, Wyer C, Angrisano F, Christophides G, Vlachou Det al., 2020,

    PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector

    , Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 7363-7373, ISSN: 0027-8424

    After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito–parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.
  • Journal article
    Ledesma-Amaro R, Nikel PI, Ceroni F, 2020,

    Editorial: synthetic biology-guided metabolic engineering

    , Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
  • Journal article
    Zielonka D, Witkowski G, Puch EA, Lesniczak M, Mazur-Michalek I, Isalan M, Mielcarek Met al., 2020,

    Prevalence of non-psychiatric comorbidities in pre-symptomatic and symptomatic Huntington's disease gene carriers in Poland

    , Frontiers in Medicine, Vol: 7, ISSN: 2296-858X

    Huntington's disease (HD) is monogenic neurodegenerative disorder caused by CAG expansions within the Huntingtin gene (Htt); it has a prevalence of 1 in 10,000 worldwide and is invariably fatal. Typically, healthy individuals have fewer than 35 CAG repeats, while the CAG expansions range from 36 to ~200 in HD patients. The hallmark of HD is neurodegeneration, especially in the striatal nuclei, basal ganglia and cerebral cortex, leading to neurological symptoms that involve motor, cognitive, and psychiatric events. However, HD is a complex disorder that may also affect peripheral organs, so it is possible that HD patients could be affected by comorbidities. Hence, we investigated the prevalence of comorbid conditions in HD patients (pre-symptomatic and symptomatic groups) and compared the frequency of those conditions to a control group. Our groups represent 65% of HD gene carriers registered in Poland. We identified 8 clusters of comorbid conditions in both HD groups, namely: musculoskeletal, allergies, cardiovascular, neurological, gastrointestinal, thyroid, psychiatric, and ophthalmologic. We found that HD patients have a significantly higher percentage of co-existing conditions in comparison to the control group. Among the 8 clusters of diseases, musculoskeletal, psychiatric, and cardiovascular events were significantly more frequent in both pre- and symptomatic HD patients, while neurological and gastrointestinal clusters showed significantly higher occurrence in the HD symptomatic group. A greater recognition of comorbidity in HD might help to better understand health outcomes and improve clinical management.
  • Journal article
    Trinugroho J, Bečková M, Shao S, Yu J, Zhao Z, Murray JW, Sobotka R, Komenda J, Nixon PJet al., 2020,

    Chlorophyll f synthesis by a super-rogue photosystem II complex

    , Nature Plants, Vol: 6, Pages: 238-244, ISSN: 2055-0278

    Certain cyanobacteria synthesize chlorophyll molecules (Chl d and Chl f) that absorb in the far-red region of the solar spectrum, thereby extending the spectral range of photosynthetically active radiation1,2. The synthesis and introduction of these far-red chlorophylls into the photosynthetic apparatus of plants might improve the efficiency of oxygenic photosynthesis, especially in far-red enriched environments, such as in the lower regions of the canopy3. Production of Chl f requires the ChlF subunit, also known as PsbA4 (ref. 4) or super-rogue D1 (ref. 5), a paralogue of the D1 subunit of photosystem II (PSII) which, together with D2, bind cofactors involved in the light-driven oxidation of water. Current ideas suggest that ChlF oxidizes Chl a to Chl f in a homodimeric ChlF reaction centre (RC) complex and represents a missing link in the evolution of the heterodimeric D1/D2 RC of PSII (refs. 4,6). However, unambiguous biochemical support for this proposal is lacking. Here, we show that ChlF can substitute for D1 to form modified PSII complexes capable of producing Chl f. Remarkably, mutation of just two residues in D1 converts oxygen-evolving PSII into a Chl f synthase. Overall, we have identified a new class of PSII complex, which we term ‘super-rogue’ PSII, with an unexpected role in pigment biosynthesis rather than water oxidation.
  • Journal article
    McCarty NS, Graham AE, Studená L, Ledesma-Amaro Ret al., 2020,

    Multiplexed CRISPR technologies for gene editing and transcriptional regulation

    , Nature Communications, Vol: 11, ISSN: 2041-1723

    Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.
  • Journal article
    Papathanasiou MM, Kontoravdi C, 2020,

    Engineering challenges in therapeutic protein product and process design

    , Current Opinion in Chemical Engineering, Vol: 27, Pages: 81-88, ISSN: 2211-3398

    Biologics represent the fastest growing sector of the pharmaceutical industry, yet their manufacture lags significantly behind that of small molecule drugs. This paper discusses the main product-related and process-related challenges during the development and production of therapeutic proteins, with particular focus on product heterogeneity and process monitoring and analytics. Emphasis is placed on novel contributions from the field of computational research that aim to enable the application of model-based process control strategies or are working towards the development of a digital twin of bioprocesses. Lastly, we review promising developments in the paradigm shift from batch to continuous processing.
  • Journal article
    Riangrungroj P, Polizzi KM, 2020,

    BeQuIK (Biosensor Engineered Quorum Induced Killing): designer bacteria for destroying recalcitrant biofilms.

    , Microbial Biotechnology, Vol: 13, Pages: 311-314, ISSN: 1751-7915

    This opinion piece describes a new design for the remediation of recalcitrant biofilms. It builds on previous work to develop engineered E. coli that recognize quorum sensing signals from pathogens in a biofilm and secrete toxins in response. To solve the challenge of dilute signalling molecules, we propose to use nanobodies and enzymes displayed on the surface of the cells to localize them to the biofilm and degrade the extracellular polymeric substances, thus creating a solution with better 'seek and destroy' capabilities.
  • Journal article
    Stach L, Morgan RM, Makhlouf L, Douangamath A, von Delft F, Zhang X, Freemont PSet al., 2020,

    Crystal structure of the catalytic D2 domain of the AAA+ ATPase p97 reveals a putative helical split-washer-type mechanism for substrate unfolding

    , FEBS Letters, Vol: 594, Pages: 933-943, ISSN: 0014-5793

    Several pathologies have been associated with the AAA+ ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High‐resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high‐resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P61 symmetry suggests a mechanism for substrate unfolding by p97.
  • Journal article
    Rodgers FH, Cai JA, Pitaluga AN, Mengin-Lecreulx D, Gendrin M, Christophides GKet al., 2020,

    Functional analysis of the three major PGRPLC isoforms in the midgut of the malaria mosquito Anopheles coluzzii

    , INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, Vol: 118, ISSN: 0965-1748
  • Journal article
    Fasulo B, Meccariello A, Morgan M, Borufka C, Papathanos PA, Windbichler Net al., 2020,

    A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    , PLOS GENETICS, Vol: 16, ISSN: 1553-7404
  • Journal article
    Spice AJ, Aw R, Bracewell DG, Polizzi KMet al., 2020,

    Synthesis and assembly of Hepatitis B virus-like particles in a Pichia pastoris cell-free system

    , Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185

    Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.
  • Journal article
    Jacobsen IH, Ledesma-Amaro R, Martinez JL, 2020,

    Recombinant β-carotene production by yarrowia lipolytica - assessing the potential of micro-scale fermentation analysis in cell factory design and bioreaction optimization

    , Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185

    The production of β-carotene has become increasingly interesting within the biotechnological industry due to a rising demand for safer and more natural colorants, nutritional supplements, and antioxidants. A recent study has described the potential of Yarrowia lipolytica as a β-carotene-producing cell factory, reporting the highest titer of recombinant β-carotene produced to date. Finding the best conditions to maximize production and scaling up the process to full scale, a costly and time-consuming process, it is often a bottleneck in biotechnology. In this work, we explored the benefits of using micro-fermentation equipment to significantly reduce the time spent on design and optimization of bioreaction conditions, especially in the early stages of process development. In this proof-of-concept study, a β-carotene producing Y. lipolytica strain was tested in micro-fermentations partly to assess the robustness of the cell factory design and partly to perform media optimization. The medium optimization led us to an improvement of up to 50% in the yield of β-carotene production in the best of the conditions. Overall, the micro-fermentation system had a high degree of reliability in all tests.
  • Journal article
    Gowers G, Chee S, Bell D, Suckling L, Kern M, Tew D, McClymont D, Ellis Tet al., 2020,

    Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening

    , Nature Communications, Vol: 11, ISSN: 2041-1723

    Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.
  • Journal article
    Wu Y, Chen T, Liu Y, Tian R, Lv X, Li J, Du G, Chen J, Ledesma-Amaro R, Liu Let al., 2020,

    Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis

    , Nucleic Acids Research, Vol: 48, Pages: 996-1009, ISSN: 0305-1048

    Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.
  • Journal article
    Wen Z, Ledesma-Amaro R, Lu M, Jin M, Yang Set al., 2020,

    Metabolic engineering of clostridium cellulovorans to improve butanol production by consolidated bioprocessing.

    , ACS Synthetic Biology, Vol: 9, Pages: 304-315, ISSN: 2161-5063

    Clostridium cellulovorans DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, C. cellulovorans was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene (ter), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating xylR (Clocel_0594) and araR (Clocel_1253), as well as overexpressing xylT (CA_C1345), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of n-butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing.
  • Journal article
    Park Y-K, Ledesma-Amaro R, Nicaud J-M, 2020,

    De novo biosynthesis of odd-chain fatty acids in yarrowia lipolytica enabled by modular pathway engineering

    , Frontiers in Bioengineering and Biotechnology, Vol: 7, Pages: 1-11, ISSN: 2296-4185

    Microbial oils are regarded as promising alternatives to fossil fuels as concerns over environmental issues and energy production systems continue to mount. Odd-chain fatty acids (FAs) are a type of valuable lipid with various applications: they can serve as biomarkers, intermediates in the production of flavor and fragrance compounds, fuels, and plasticizers. Microorganisms naturally produce FAs, but such FAs are primarily even-chain; only negligible amounts of odd-chain FAs are generated. As a result, studies using microorganisms to produce odd-chain FAs have had limited success. Here, our objective was to biosynthesize odd-chain FAs de novo in Yarrowia lipolytica using inexpensive carbon sources, namely glucose, without any propionate supplementation. To achieve this goal, we constructed a modular metabolic pathway containing seven genes. In the engineered strain expressing this pathway, the percentage of odd-chain FAs out of total FAs was higher than in the control strain (3.86 vs. 0.84%). When this pathway was transferred into an obese strain, which had been engineered to accumulate large amounts of lipids, odd-chain fatty acid production was 7.2 times greater than in the control (0.05 vs. 0.36 g/L). This study shows that metabolic engineering research is making progress toward obtaining efficient cell factories that produce odd-chain FAs.
  • Journal article
    Sattayawat P, Yunus IS, Jones PR, 2020,

    Bioderivatization as a concept for renewable production of chemicals that are toxic or poorly soluble in the liquid phase

    , Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 1404-1413, ISSN: 0027-8424

    Bio-based production technologies may complement or replace petroleum-based production of chemicals, but they face a number of technical challenges, including product toxicity and/or water insolubility. Plants and microorganisms naturally biosynthesize chemicals that often are converted into derivatives with reduced toxicity or enhanced solubility. Inspired by this principle, we propose a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As proof of principle, the effects of hydrophobic (e.g., esterification) and hydrophilic (e.g., glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic and poorly soluble chemical, 1-octanol, were evaluated in Escherichia coli and Synechocystis sp. PCC 6803. The 1-octanol pathway was first optimized to reach product titers at which the host displayed symptoms of toxicity. Solvent overlay used to capture volatile products partially masked product toxicity. Regardless of whether solvent overlay was used, most strains with bioderivatization had a higher molar product titer and product yield, as well as improved cellular growth and glucose consumption, compared with strains without bioderivatization. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. Interestingly, in several combinations of genotype/induction strength, bioderivatization had a positive effect on productivity without any apparent effect on growth. We attribute this to enhanced product solubility in the aqueous or solvent fraction of the bioreactor liquid phase (depending on the derivative and medium used), with consequent enhanced product removal. Overall, under most conditions, a benefit of bioproduction was observed, and the bioderivatization strategy could be considered for other similar chemicals as well.
  • Journal article
    Kopniczky MB, Canavan C, McClymont DW, Crone MA, Suckling L, Goetzmann B, Siciliano V, MacDonald JT, Jensen K, Freemont PSet al., 2020,

    Cell-free protein synthesis as a prototyping platform for mammalian synthetic biology

    , ACS Synthetic Biology, Vol: 9, Pages: 144-156, ISSN: 2161-5063

    The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.
  • Journal article
    Yunus IS, Palma A, Trudeau DL, Tawfik DS, Jones PRet al., 2020,

    Methanol-free biosynthesis of fatty acid methyl ester (FAME) in <i>Synechocystis</i> sp. PCC 6803

    , METABOLIC ENGINEERING, Vol: 57, Pages: 217-227, ISSN: 1096-7176

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