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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
    Gowers G-OF, Cameron SJS, Perdones-Montero A, Bell D, Chee SM, Kern M, Tew D, Ellis T, Takats Zet al., 2019,

    Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry

    , ACS Synthetic Biology, Vol: 8, Pages: 2566-2575, ISSN: 2161-5063

    Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) - a form of ambient laser desorption ionisation mass spectrometry - to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.

  • Conference paper
    Webb AJ, Kelwick R, Wang Y, Heliot A, Templeton MR, Freemont PSet al., 2019,

    AL-PHA beads: bioplastic-bsaed protease biosensors for global health

    , British Society for Parasitology Autumn Symposium, Belfast, UK
  • Journal article
    Fasulo B, Meccariello A, Morgan M, Borufka C, Papathanos PA, Windbichler Net al., 2019,

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

    <jats:p>Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established<jats:italic>Drosophila</jats:italic>models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion - “X-shredding” and “X-meddling” - and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species.</jats:p>

  • Journal article
    Dahalan FA, Churcher TS, Windbichler N, Lawniczak MKNet al., 2019,

    The male mosquito contribution towards malaria transmission: Mating influences the Anopheles female midgut transcriptome and increases female susceptibility to human malaria parasites.

    , PLoS Pathogens, Vol: 15, Pages: 1-19, ISSN: 1553-7366

    Mating causes dramatic changes in female physiology, behaviour, and immunity in many insects, inducing oogenesis, oviposition, and refractoriness to further mating. Females from the Anopheles gambiae species complex typically mate only once in their lifetime during which they receive sperm and seminal fluid proteins as well as a mating plug that contains the steroid hormone 20-hydroxyecdysone. This hormone, which is also induced by blood-feeding, plays a major role in activating vitellogenesis for egg production. Here we show that female Anopheles coluzzii susceptibility to Plasmodium falciparum infection is significantly higher in mated females compared to virgins. We also find that mating status has a major impact on the midgut transcriptome, detectable only under sugar-fed conditions: once females have blood-fed, the transcriptional changes that are induced by mating are likely masked by the widespread effects of blood-feeding on gene expression. To determine whether increased susceptibility to parasites could be driven by the additional 20E that mated females receive from males, we mimicked mating by injecting virgin females with 20E, finding that these females are significantly more susceptible to human malaria parasites than virgin females injected with the control 20E carrier. Further RNAseq was carried out to examine whether the genes that change upon 20E injection in the midgut are similar to those that change upon mating. We find that 79 midgut-expressed genes are regulated in common by both mating and 20E, and 96% (n = 76) of these are regulated in the same direction (up vs down in 20E/mated). Together, these findings show that male Anopheles mosquitoes induce changes in the female midgut that can affect female susceptibility to P. falciparum. This implies that in nature, males might contribute to malaria transmission in previously unappreciated ways, and that vector control strategies that target males may have additional benefits towards reducing transm

  • Journal article
    Gowers G-O, Vince O, Charles J-H, Klarenberg I, Ellis T, Edwards Aet al., 2019,

    Entirely off-grid and solar-powered DNA sequencing of microbial communities during an ice cap traverse expedition

    , Genes, Vol: 10, Pages: 1-10, ISSN: 2073-4425

    Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth’s surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions. While remote research stations have significantly lowered the barrier to studying the microbial communities on icecaps, their use has led to a bias for data collection in the near vicinity of these institutions. Here, miniaturisation of a DNA sequencing lab suitable for off-grid metagenomic studies is demonstrated. Using human power alone, this lab was transported across Europe’s largest ice cap (Vatnajökull, Iceland) by ski and sledge. After 11 days of unsupported polar-style travel, a metagenomic study of a geothermal hot spring gorge was conducted on the remote northern edge of the ice cap. This tent-based metagenomic study resulted in over 24 h of Nanopore sequencing, powered by solar power alone. This study demonstrates the ability to conduct DNA sequencing in remote locations, far from civilised resources (mechanised transport, external power supply, internet connection, etc.), whilst greatly reducing the time from sample collection to data acquisition.

  • Journal article
    Lai H-E, Canavan C, Cameron L, Moore S, Danchenko M, Kuiken T, Sekeyová Z, Freemont PSet al., 2019,

    Synthetic biology and the United Nations

    , Trends in Biotechnology, Vol: 37, Pages: 1146-1151, ISSN: 0167-7799

    Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).

  • Journal article
    Jiménez A, Muñoz-Fernández G, Ledesma-Amaro R, Buey RM, Revuelta JLet al., 2019,

    One-vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

    , Microbial Biotechnology, Vol: 12, Pages: 1293-1301, ISSN: 1751-7915

    The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low-cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high-added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one-vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker-less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker-less manner.

  • Journal article
    Ostrov N, Beal J, Ellis T, Gordon DB, Karas BJ, Lee HH, Lenaghan SC, Schloss JA, Stracquadanio G, Trefzer A, Bader JS, Church GM, Coelho CM, Efcavitch JW, Güell M, Mitchell LA, Nielsen AAK, Peck B, Smith AC, Stewart CN, Tekotte Het al., 2019,

    Technological challenges and milestones for writing genomes.

    , Science, Vol: 366, Pages: 310-312, ISSN: 0036-8075

    Engineering biology with recombinant DNA, broadly called synthetic biology, has progressed tremendously in the last decade, owing to continued industrialization of DNA synthesis, discovery and development of molecular tools and organisms, and increasingly sophisticated modeling and analytic tools. However, we have yet to understand the full potential of engineering biology because of our inability to write and test whole genomes, which we call synthetic genomics. Substantial improvements are needed to reduce the cost and increase the speed and reliability of genetic tools. Here, we identify emerging technologies and improvements to existing methods that will be needed in four major areas to advance synthetic genomics within the next 10 years: genome design, DNA synthesis, genome editing, and chromosome construction (see table). Similar to other large-scale projects for responsible advancement of innovative technologies, such as the Human Genome Project, an international, cross-disciplinary effort consisting of public and private entities will likely yield maximal return on investment and open new avenues of research and biotechnology.

  • Journal article
    McFarlane C, Shah N, Kabasakal B, Echeverria B, Cotton C, Bubeck D, Murray Jet al., 2019,

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

    , Proceedings of the National Academy of Sciences of USA, Vol: 116, Pages: 20984-20990, ISSN: 0027-8424

    Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) are essential Calvin-Benson cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyses the reduction step of the CB cycle with NADPH to produce the sugar, glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are co-regulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of Calvin-Benson cycle regulation by CP12 is unknown. Here we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologues, we solve crystal structures of GAPDH with different cofactors and CP12 bound, 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, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.

  • Journal article
    Freemont P, 2019,

    Synthetic biology industry - Data-driven design is creating new opportunities in biotechnology.

    , Emerging Topics in Life Sciences, Vol: 3, Pages: 651-657, ISSN: 2397-8554

    Synthetic biology is a rapidly emerging interdisciplinary research field that is primarily built upon foundational advances in molecular biology combined with engineering design. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies. This has spurned a rapid growth in start-up companies and the new synthetic biology industry is growing rapidly, with start-up companies receiving ~$6.1B investment since 2015 and a global synthetic biology market value estimated to be $14B by 2026. Many of the new start-upscan be grouped within a multi-layer ‘technology stack’. The ‘stack’ comprises a number of technology layers which together can be applied to a diversity of new biotechnology applications like consumer biotechnology products and living therapies. The ‘stack’ also enables new commercial opportunities and value chains similar to the software design and manufacturing revolution of the 20th century. However, synthetic biology industry is at a crucial point, as it now requires recognisable commercial successes in order for the industry to expand and scale, in terms of investment and companies. However, such expansion may directly challenge the ethos of synthetic biology, in terms of open technology sharing and democratisation, which could by accident lead to multi-national corporations and technology monopolies similar to the existing biotechnology/biopharma industry.

  • Journal article
    Giese B, Friess JL, Barton NH, Messer PW, Debarre F, Schetelig MF, Windbichler N, Meimberg H, Boete Cet al., 2019,

    Gene Drives: Dynamics and Regulatory Matters-A Report from the Workshop "Evaluation of Spatial and Temporal Control of Gene Drives," April 4-5, 2019, Vienna

    , BIOESSAYS, Vol: 41, ISSN: 0265-9247
  • Journal article
    Wood TE, Howard SA, Forster A, Nolan LM, Manoli E, Bullen NP, Yau HCL, Hachani A, Hayward RD, Whitney JC, Vollmer W, Freemont PS, Filloux Aet al., 2019,

    The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology

    , Cell Reports, Vol: 29, Pages: 187-201.e7, ISSN: 2211-1247

    The type VI secretion system (T6SS) is crucialin interbacterial competition and is avirulence determinant ofmany Gram-negative bacteria. Several T6SS effectorsarecovalently fused to secreted T6SS structural components such asthe VgrG spike for delivery into target cells.In Pseudomonas aeruginosa, theVgrG2b effector waspreviously proposedto mediatebacterial internalisation into eukaryotic cells. In this work, wefind that the VgrG2b C-terminal domain(VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein.We resolve thestructure of VgrG2bC-ter and confirm it is a member ofthezinc-metallopeptidasefamily of enzymes. We show that this effector causesmembrane blebbing atmidcell, whichsuggests a distincttype of T6SS-mediated growthinhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Ourstudyintroduces a further effector family to the T6SS arsenaland demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.

  • Journal article
    Meccariello A, Salvemini M, Primo P, Hall B, Koskiniot P, Dalikova M, Gravina A, Gucciar MA, Forlenza F, Gregoriou ME, Ippolito D, Monti SM, Petrella V, Perrotta MM, Schmeing S, Ruggiero A, Scolari F, Giordano E, Tsoumani KT, Marec F, Windbichler N, Nagaraju J, Arunkumar KP, Bourtzis K, Mathiopoulos KD, Ragoussis J, Vitagliano L, Tu Z, Papathanos PA, Robinson MD, Saccone Get al., 2019,

    Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests

    , Sciecnce, Vol: 365, Pages: 1457-1460, ISSN: 0036-8075

    In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.

  • Journal article
    Scholes N, Schnoerr D, Isalan M, Stumpf Met al., 2019,

    A comprehensive network atlas reveals that Turing patterns are common but not robust

    , Cell Systems, Vol: 9, Pages: 243-257.e4, ISSN: 2405-4712

    Turing patterns (TPs) underlie many fundamental developmental processes, but they operate over narrow parameter ranges, raising the conundrum of how evolution can ever discover them. Here we explore TP design space to address this question and to distill design rules. We exhaustively analyze 2- and 3-node biological candidate Turing systems, amounting to 7,625 networks and more than 3 × 10^11 analyzed scenarios. We find that network structure alone neither implies nor guarantees emergent TPs. A large fraction (>61%) of network design space can produce TPs, but these are sensitive to even subtle changes in parameters, network structure, and regulatory mechanisms. This implies that TP networks are more common than previously thought, and evolution might regularly encounter prototypic solutions. We deduce compositional rules for TP systems that are almost necessary and sufficient (96% of TP networks contain them, and 92% of networks implementing them produce TPs). This comprehensive network atlas provides the blueprints for identifying natural TPs and for engineering synthetic systems.

  • Journal article
    Kelwick RJR, Ricci L, Chee SM, Bell D, Webb A, Freemont Pet al., 2019,

    Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

    , Synthetic Biology, Vol: 3, ISSN: 2397-7000

    The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimise several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimising PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customised our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimise in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ∼50%. In cell-free transcription-translation prototyping reactions, GC-MS quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ±0.39 µM), C104 ΔPhaC_C319A (4.62 ±1.31 µM) and C101 ΔPhaC_C319A (2.65 ±1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription-translation reactions produced greater yields of 3HB (32.87 ±6.58 µM) and these reactions were also used to characterise a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimising PHAs production.

  • Journal article
    Kitney R, Adeogun M, Fujishima Y, Goni-Moreno A, Johnson R, Maxon M, Steedman S, Ward S, Winickoff D, Philp Jet al., 2019,

    Enabling the Advanced Bioeconomy through Public Policy Supporting Biofoundries and Engineering Biology

    , TRENDS IN BIOTECHNOLOGY, Vol: 37, Pages: 917-920, ISSN: 0167-7799
  • Conference paper
    Hurault G, Tanaka RJ, 2019,

    A Bayesian machine learning model to identify patient-specific dynamic responses to eczema treatments

    , 49th Annual Meeting of the European-Society-for-Dermatological-Research (ESDR), Publisher: ELSEVIER SCIENCE INC, Pages: S239-S239, ISSN: 0022-202X
  • Journal article
    Deng J, Chen C, Gu Y, Lv X, Liu Y, Li J, Ledesma-Amaro R, Du G, Liu Let al., 2019,

    Creating an in vivo bifunctional gene expression circuit through an aptamer-based regulatory mechanism for dynamic metabolic engineering in Bacillus subtilis.

    , Metabolic Engineering, Vol: 55, Pages: 179-190, ISSN: 1096-7176

    Aptamer-based regulatory biosensors can dynamically regulate the expression of target genes in response to ligands and could be used in dynamic metabolic engineering for pathway optimization. However, the existing aptamer-ligand biosensors can only function with non-complementary DNA elements that cannot replicate in growing cells. Here, we construct an aptamer-based synthetic regulatory circuit that can dynamically upregulate and downregulate the expression of target genes in response to the ligand thrombin at transcriptional and translational levels, respectively, and further used this system to dynamically engineer the synthesis of 2'-fucosyllactose (2'-FL) in Bacillus subtilis. First, we demonstrated the binding of ligand molecule thrombin with the aptamer can induce the unwinding of fully complementary double-stranded DNA. Based on this finding, we constructed a bifunctional gene expression regulatory circuit using ligand thrombin-bound aptamers. The expression of the reporter gene ranged from 0.084- to 48.1-fold. Finally, by using the bifunctional regulatory circuit, we dynamically upregulated the expression of key genes fkp and futC and downregulated the expression of gene purR, resulting in the significant increase of 2'-FL titer from 24.7 to 674 mg/L. Compared with the other pathway-specific dynamic engineering systems, here the constructed aptamer-based regulatory circuit is independent of pathways, and can be generally used to fine-tune gene expression in other microbes.

  • Journal article
    Bartasun P, Prandi N, Storch M, Aknin Y, Bennett M, Palma A, Baldwin G, Sakuragi Y, Jones PR, Rowland Jet al., 2019,

    The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803

    , PEERJ, Vol: 7, ISSN: 2167-8359

    Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced into Synechocystis sp. PCC 6803. We characteriseda new set of ribosome binding site sequences in Synechocystis sp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested con

  • Journal article
    Bruder S, Moldenhauer EJ, Lemke RD, Ledesma-Amaro R, Kabisch Jet al., 2019,

    Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica.

    , Biotechnol Biofuels, Vol: 12, Pages: 1-13, ISSN: 1754-6834

    Background: Oleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches. Results: To demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed. Conclusions: Oleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route t

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