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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
    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.

  • 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
    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
    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.

  • 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.

  • Conference paper
    Thaore V, Moore S, Polizzi K, Freemont P, Shah N, Kontoravdi Cet al., 2018,

    Techno-economic evaluation of a cell-free syntheticbiochemistry route for raspberry ketone production atindustrial scale

    , Vaishali Thaore
  • Journal article
    Oling D, Lawenius L, Shaw W, Clark S, Kettleborough R, Ellis T, Larsson N, Wigglesworth Met al., 2018,

    Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling

    , ACS SYNTHETIC BIOLOGY, Vol: 7, Pages: 2317-2321, ISSN: 2161-5063
  • Journal article
    Gamboa-Melendez H, Larroude M, Park YK, Trebul P, Nicaud J-M, Ledesma-Amaro Ret al., 2018,

    Synthetic Biology to Improve the Production of Lipases and Esterases (Review).

    , Methods Mol Biol, Vol: 1835, Pages: 229-242

    Synthetic biology is an emergent field of research whose aim is to make biology an engineering discipline, thus permitting to design, control, and standardize biological processes. Synthetic biology is therefore expected to boost the development of biotechnological processes such as protein production and enzyme engineering, which can be significantly relevant for lipases and esterases.

  • Journal article
    Yu J, Knoppova J, Michoux F, Bialek W, Cota Segura E, Shukla M, Straskova A, Aznar G, Sobotka R, Komenda J, Murray J, Nixon PJet al., 2018,

    Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein

    , Proceedings of the National Academy of Sciences, Vol: 115, Pages: E7824-E7833, ISSN: 0027-8424

    Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved “Arg patch” on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

  • Journal article
    Ceroni F, Ellis T, 2018,

    The challenges facing synthetic biology in eukaryotes

    , Nature Reviews Molecular Cell Biology, Vol: 19, Pages: 481-482, ISSN: 1471-0072

    Synthetic biology is maturing into a true engineering discipline for model microorganisms, but remains far from straightforward for most eukaryotes. Here, we outline the key challenges facing those trying to engineer biology across eukaryota and suggest areas of focus that will aid future progress.

  • Journal article
    Kylilis N, Tuza ZA, Stan G, Polizzi KMet al., 2018,

    Tools for engineering coordinated system behaviour in synthetic microbial consortia

    , Nature Communications, Vol: 9, Pages: 1-9, ISSN: 2041-1723

    Advancing synthetic biology to the multicellular level requires the development of multiple cell-to-cell communication channels that propagate information with minimal signal interference. The development of quorum-sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on cognate acyl-homoserine lactone (AHL)/transcription factor pairs, while the use of non-cognate pairs as a design feature has received limited attention. Here, we demonstrate a large library of AHL-receiver devices, with all cognate and non-cognate chemical signal interactions quantified, and we develop a software tool that automatically selects orthogonal communication channels. We use this approach to identify up to four orthogonal channels in silico, and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels is an enabling step that facilitates the design of synthetic consortia for applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation and spatial organisation.

  • Journal article
    Tanaka G, Dominguez-Huttinger E, Christodoulides P, Kazuyuki A, Tanaka RJet al., 2018,

    Bifurcation analysis of a mathematical model of atopic dermatitis to determine patient-specific effects of treatments on dynamic phenotypes

    , Journal of Theoretical Biology, Vol: 448, Pages: 66-79, ISSN: 0022-5193

    Atopic dermatitis (AD) is a common inflammatory skin disease, whose incidence is currently increasing worldwide. AD has a complex etiology, involving genetic, environmental, immunological, and epidermal factors, andits pathogenic mechanisms have not yet been fully elucidated. Identificationof AD risk factors and systematic understanding of their interactions arerequired for exploring effective prevention and treatment strategies for AD.We recently developed a mathematical model for AD pathogenesis to clarifymechanisms underlying AD onset and progression. This model describes adynamic interplay between skin barrier, immune regulation, and environmental stress, and reproduced four types of dynamic behaviour typically observed in AD patients in response to environmental triggers. Here, we analyse bifurcations of the model to identify mathematical conditions for the system to demonstrate transitions between different types of dynamic behaviour that reflect respective severity of AD symptoms. By mathematically modelling effects of topical application of antibiotics, emollients, corticosteroids, and their combinations with different application schedules and doses, bifurcation analysis allows us to mathematically evaluate effects of the treatments on improving AD symptoms in terms of the patients' dynamic behaviour. The mathematical method developed in this study can be used to explore and improve patient-specific personalised treatment strategies to control AD symptoms.

  • Journal article
    Goey CH, Alhuthali S, Kontoravdi K, 2018,

    Host cell protein removal from biopharmaceutical preparations: toward the implementation of quality by design

    , Biotechnology Advances, Vol: 36, Pages: 1223-1237, ISSN: 0734-9750

    Downstream processing of protein products of mammalian cell culture currently accounts for the largest fraction of the total production cost. A major challenge is the removal of host cell proteins, which are cell-derived impurities. Host cell proteins are potentially immunogenic and can compromise product integrity during processing and hold-up steps. There is an increasing body of evidence that the type of host cell proteins present in recombinant protein preparations is a function of cell culture conditions and handling of the harvest cell culture fluid. This, in turn, can affect the performance of downstream purification steps as certain species are difficult to remove and may require bespoke process solutions. Herein, we review recent research on the interplay between upstream process conditions, host cell protein composition and their downstream removal in antibody production processes, identifying opportunities for increasing process understanding and control. We further highlight advances in analytical and computational techniques that can enable the application of quality by design.

  • Journal article
    Liu D, Mannan AA, Han Y, Oyarzun DA, Zhang Fet al., 2018,

    Dynamic metabolic control: towards precision engineering of metabolism

    , Journal of Industrial Microbiology and Biotechnology, Vol: 45, Pages: 535-543, ISSN: 1367-5435

    Advances in metabolic engineering have led to the synthesis of a wide variety of valuable chemicals in microorganisms. The key to commercializing these processes is the improvement of titer, productivity, yield, and robustness. Traditional approaches to enhancing production use the “push–pull-block” strategy that modulates enzyme expression under static control. However, strains are often optimized for specific laboratory set-up and are sensitive to environmental fluctuations. Exposure to sub-optimal growth conditions during large-scale fermentation often reduces their production capacity. Moreover, static control of engineered pathways may imbalance cofactors or cause the accumulation of toxic intermediates, which imposes burden on the host and results in decreased production. To overcome these problems, the last decade has witnessed the emergence of a new technology that uses synthetic regulation to control heterologous pathways dynamically, in ways akin to regulatory networks found in nature. Here, we review natural metabolic control strategies and recent developments in how they inspire the engineering of dynamically regulated pathways. We further discuss the challenges of designing and engineering dynamic control and highlight how model-based design can provide a powerful formalism to engineer dynamic control circuits, which together with the tools of synthetic biology, can work to enhance microbial production.

  • Conference paper
    Hurault G, Roekevisch E, Szegedi K, Kezic S, Spuls PI, Middelkamp-Hup MA, Tanaka RJet al., 2018,

    Predicting short- and long-term outcomes of a systemic therapy for atopic dermatitis using machine learning methods

    , 10th George Rajka International Symposium on Atopic Dermatitis, Publisher: Wiley, Pages: E17-E18, ISSN: 1365-2133
  • Journal article
    Nash A, Urdaneta GM, K Beaghton A, Hoermann A, Aris Papathanos P, Christophides GK, Windbichler Net al., 2018,

    Integral Gene Drives: an “operating system” for population replacement

    <jats:title>Abstract</jats:title><jats:p>First generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population while hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.</jats:p>

  • Journal article
    Beal J, Haddock-Angelli T, Baldwin G, Gershater M, Dwijayanti A, Storch M, de Mora K, Lizarazo M, Rettberg Ret al., 2018,

    Quantification of bacterial fluorescence using independent calibrants

    , PLoS ONE, Vol: 13, ISSN: 1932-6203

    Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.

  • Conference paper
    Giannari AG, van Logtestijn MDA, Christodoulides P, Konishi K, Tanaka RJet al., 2018,

    Model Predictive Control for Designing Proactive Therapy of Atopic Dermatitis

    , European Control Conference (ECC), Publisher: IEEE, Pages: 2387-2392
  • Journal article
    Enrico Bena C, Grob A, Isalan M, Bosia C, Ceroni Fet al., 2018,

    Commentary: Synthetic Addiction Extends the Productive Life Time of Engineered Escherichia coli Populations

    , Frontiers in Bioengineering and Biotechnology, Vol: 6, ISSN: 2296-4185

    A commentary on Synthetic addiction extends the productive life time of engineered Escherichia coli populations by Rugbjerg, P., Sarup-Lytzen, K., Nagy, M., and Sommer, M. O. A. (2018). Proc. Natl. Acad. Sci. U.S.A. 115, 2347–2352. doi: 10.1073/pnas.1718622115Bioproduction is the process of producing added-value chemicals on large-scale using cells as biological factories. Cellular burden represents a significant problem in the scaling of fermentation processes from proof-of-concept to long-term cultures, as the load of heterologous gene expression and depletion of the cell intracellular resources cause unpredictable cellular physiological changes that can lead to decreased growth and lower production yields (Borkowski et al., 2016; Liu et al., 2018). One possible cause of the observed decreased bioproduct recovery in many bioprocessing applications is the accumulation of mutations in the employed genetic program. These mutations often lead to loss of production and rise of non-producing populations that grow better and easily overtake the growth of producing cells (Rugbjerg et al., 2018b).In a recent paper in PNAS, Rugbjerg et al. (2018b) developed a strategy to limit the enrichment of non-producing cell populations in bioproduction-employed cell cultures by placing the genes for key growth intermediates under the control of a promoter responsive to the bioproduct being made. This strategy known as product addiction was tested in E. coli engineered to produce mevalonic acid in long-term cultivations (Figure 1).

  • Journal article
    Park YK, Dulermo T, Ledesma Amaro R, Nicaud J-Met al., 2018,

    Optimization of odd chain fatty acid production by Yarrowia lipolytica

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

    Background:Odd chain fatty acids (odd FAs) have a wide range of applications in therapeutic and nutritional industries, as well as in chemical industries including biofuel. Yarrowia lipolytica is an oleaginous yeast considered a preferred microorganism for the production of lipid-derived biofuels and chemicals. However, it naturally produces negligible amounts of odd chain fatty acids.Results:The possibility of producing odd FAs using Y. lipolytica was investigated. Y. lipolytica wild-type strain was shown able to grow on weak acids; acetate, lactate, and propionate. Maximal growth rate on propionate reached 0.24 ± 0.01 h−1 at 2 g/L, and growth inhibition occurred at concentration above 10 g/L. Wild-type strain accumulated lipids ranging from 7.39 to 8.14% (w/w DCW) depending on the carbon source composition, and odd FAs represented only 0.01–0.12 g/L. We here proved that the deletion of the PHD1 gene improved odd FAs production, which reached a ratio of 46.82% to total lipids. When this modification was transferred to an obese strain, engineered for improving lipid accumulation, further increase odd FAs production reaching a total of 0.57 g/L was shown. Finally, a fed-batch co-feeding strategy was optimized for further increase odd FAs production, which generated 0.75 g/L, the best production described so far in Y. lipolytica.Conclusions:A Y. lipolytica strain able to accumulate high level of odd chain fatty acids, mainly heptadecenoic acid, has been successfully developed. In addition, a fed-batch co-feeding strategy was optimized to further improve lipid accumulation and odd chain fatty acid content. These lipids enriched in odd chain fatty acid can (1) improve the properties of the biodiesel generated from Y. lipolytica lipids and (2) be used as renewable source of odd chain fatty acid for industrial applications. This work paves the way for further improvements in odd chain fatty acids and fatty acid-derived compound production.

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