Quorum sensing in synthetic biology: A review

Whiteley, 2017, Progress in and promise of bacterial quorum sensing research, Nature, 551, 313, 10.1038/nature24624 Mukherjee, 2019, Bacterial quorum sensing in complex and dynamically changing environments, Nat Rev Microbiol, 17, 371, 10.1038/s41579-019-0186-5 Stephens, 2020, Synthetic biology for manipulating quorum sensing in microbial consortia, Trends Microbiol, 28, 633, 10.1016/j.tim.2020.03.009 Miller, 2001, Quorum sensing in bacteria, Annu Rev Microbiol, 55, 165, 10.1146/annurev.micro.55.1.165 Sharma, 2020, Quorum sensing: its role in microbial social networking, Res Microbiol, 171, 159, 10.1016/j.resmic.2020.06.003 Engebrecht, 1983, Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri, Cell, 32, 773, 10.1016/0092-8674(83)90063-6 Bassler, 1994, Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway, Mol Microbiol, 13, 273, 10.1111/j.1365-2958.1994.tb00422.x Miller, 2001, Quorum sensing in bacteria, Annu Rev Microbiol, 55, 165, 10.1146/annurev.micro.55.1.165 Hammer, 2003, Quorum sensing controls biofilm formation in Vibrio cholerae, Mol Microbiol, 50, 101, 10.1046/j.1365-2958.2003.03688.x Barnard, 2007, Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria, Phil Trans Biol Sci, 362, 1165, 10.1098/rstb.2007.2042 De Kievit, 2000, Bacterial quorum sensing in pathogenic relationships, Infect Immun, 68, 4839, 10.1128/IAI.68.9.4839-4849.2000 Deepika, 2018, Bacterial quorum sensing in pathogenic relationships: relevance to complex signalling networks and prospective applications, 67 Marchand, 2013, Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling, Biotechnol Bioeng, 110, 3003, 10.1002/bit.24975 Kong, 2018, Designing microbial consortia with defined social interactions, Nat Chem Biol, 1 Zhou, 2017, The DSF family of quorum sensing signals: diversity, biosynthesis, and turnover, Trends Microbiol, 25, 293, 10.1016/j.tim.2016.11.013 Groenhagen, 2013, Production of bioactive volatiles by different burkholderia ambifaria strains, J Chem Ecol, 39, 892, 10.1007/s10886-013-0315-y Weisskopf, 2021, Microbial volatile organic compounds in intra-kingdom and inter-kingdom interactions, Nat Rev Microbiol, 19, 391, 10.1038/s41579-020-00508-1 Dwidar, 2019, Programmable artificial cells using histamine-responsive synthetic riboswitch, J Am Chem Soc, 141, 11103, 10.1021/jacs.9b03300 Luan, 2020, Leveraging synthetic particles for communication: from passive to active systems, Nanoscale, 12, 21015, 10.1039/D0NR05675H Du, 2020, De novo design of an intercellular signaling toolbox for multi-channel cell–cell communication and biological computation, Nat Commun, 11, 1, 10.1038/s41467-020-17993-w Youk, 2014, Secreting and sensing the same molecule allows cells to achieve versatile social behaviors, Science, 343, 10.1126/science.1242782 Billerbeck, 2018, A scalable peptide-GPCR language for engineering multicellular communication, Nat Commun, 9, 1, 10.1038/s41467-018-07610-2 Shaw, 2019, Engineering a model cell for rational tuning of GPCR signaling, Cell, 177, 782, 10.1016/j.cell.2019.02.023 Martin, 2019, Quorum sensing with pheromones, Nature Microbiology, 4, 1430, 10.1038/s41564-019-0538-y Ruiz, 2021, The architecture of a mixed fungal–bacterial biofilm is modulated by quorum-sensing signals, Environ Microbiol, 23, 2433, 10.1111/1462-2920.15444 Papenfort, 2016, Quorum sensing signal-response systems in Gram-negative bacteria, Nat Rev Microbiol, 14, 576, 10.1038/nrmicro.2016.89 Chen, 2019, Quorum sensing inhibitors from marine microorganisms and their synthetic derivatives, Mar Drugs, 17, 80, 10.3390/md17020080 Aframian, 2020, A bacterial tower of babel: quorum-sensing signaling diversity and its evolution, Annu Rev Microbiol, 74, 587, 10.1146/annurev-micro-012220-063740 Davis, 2015, Can the natural diversity of quorum-sensing advance synthetic biology?, Front. Bioeng, Biotechnol., 3, 30 Wu, 2020, Quorum sensing for population - level control of bacteria and potential therapeutic applications, Cell Mol Life Sci, 77, 1319, 10.1007/s00018-019-03326-8 Wang, 2020, Quorum sensing communication: molecularly connecting cells, their neighbors, and even devices, Ann. Rev. Chem. Biomolec. Engineerin., 11, 447, 10.1146/annurev-chembioeng-101519-124728 McCarty, 2019, Synthetic biology tools to engineer microbial communities for biotechnology, Trends Biotechnol, 37, 181, 10.1016/j.tibtech.2018.11.002 Ge, 2020, Quorum sensing system used as a tool in metabolic engineering, Biotechnol J, 15, 1, 10.1002/biot.201900360 Hartline, 2021, Dynamic control in metabolic engineering: theories, tools, and applications, Metab Eng, 63, 126, 10.1016/j.ymben.2020.08.015 Ellermann, 2020, Bacterial signaling as an antimicrobial target, Curr Opin Microbiol, 57, 78, 10.1016/j.mib.2020.08.001 Haque, 2020, Quorum sensing: a new prospect for the management of antimicrobial-resistant infectious diseases, Expert Rev Anti-infect Ther, 1 Kumar, 2020, Intrusion of bacterial quorum-sensing for antimicrobial resistance mitigation: a pharmaceutical perspective, 177, 10.1007/978-3-030-53024-2_8 Zhao, 2020, Quorum-sensing regulation of antimicrobial resistance in bacteria, Microorganisms, 8, 1, 10.3390/microorganisms8030425 Singh, 2021, Development, dynamics and control of antimicrobial-resistant bacterial biofilms: a review, Environ Chem Lett, 1 Sikdar, 2020, Quorum quenching enzymes and their effects on virulence, biofilm, and microbiomes: a review of recent advances, Expert Rev Anti-infect Ther, 18, 1221, 10.1080/14787210.2020.1794815 Carradori, 2020, Biofilm and Quorum Sensing inhibitors: the road so far, Expert Opin Ther Pat, 30, 917, 10.1080/13543776.2020.1830059 Mukherjee, 2020, Engineering controllable biofilms for biotechnological applications, Micriob. Biotechn., 14, 74, 10.1111/1751-7915.13715 Kovács, 2020, Fungal quorum-sensing molecules: a review of their antifungal effect against Candida biofilms, J. Fungi, 6, 1, 10.3390/jof6030099 Fang, 2020, Controlling biofilms using synthetic biology approaches, Biotechnol Adv, 40, 107518, 10.1016/j.biotechadv.2020.107518 Padder, 2018, Quorum sensing: a less known mode of communication among fungi, Microbiol Res, 210, 51, 10.1016/j.micres.2018.03.007 Mehmood, 2019, Fungal quorum-sensing molecules and inhibitors with potential antifungal activity: a review, Molecules, 24, 1, 10.3390/molecules24101950 Winters, 2019, Can community-based signalling behaviour in Saccharomyces cerevisiae be called quorum sensing? A critical review of the literature, FEMS Yeast Res, 19, 1, 10.1093/femsyr/foz046 Eberhard, 1981, Structural identification of autoinducer of photobacterium fischeri luciferase, Biochemistry, 20, 2444, 10.1021/bi00512a013 Engebrecht, 1984, Identification of genes and gene products necessary for bacterial bioluminescence, Proc Natl Acad Sci USA, 81, 4154, 10.1073/pnas.81.13.4154 Zhang, 2020, N-acyl-homoserine lactones (AHLs) in intertidal marsh: diversity and potential role in nitrogen cycling, Plant Soil, 454, 103, 10.1007/s11104-020-04630-0 Minogue, 2002, The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function, Mol Microbiol, 44, 1625, 10.1046/j.1365-2958.2002.02987.x Scott, 2016, Quorum sensing communication modules for microbial consortia, ACS Synth Biol, 5, 969, 10.1021/acssynbio.5b00286 Grant, 2016, Orthogonal intercellular signaling for programmed spatial behavior, Mol Syst Biol, 12, 849, 10.15252/msb.20156590 Halleran, 2017, Cell-free and in vivo characterization of Lux, Las, and Rpa quorum activation systems in E. coli, ACS Synth Biol Kylilis, 2018, Tools for engineering coordinated system behaviour in synthetic microbial consortia, Nat Commun, 9, 1, 10.1038/s41467-018-05046-2 Tekel, 2019, Engineered orthogonal quorum sensing systems for synthetic gene regulation in escherichia coli, Front. Bioeng, Biotechnol., 7, 80, 10.3389/fbioe.2019.00080 Wang, 2015, Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities, Nucleic Acids Res, 43, 1955, 10.1093/nar/gku1388 Shong, 2013, Engineering the esaR promoter for tunable quorum sensing-dependent gene expression, ACS Synth Biol, 2, 568, 10.1021/sb4000433 Collins, 2005, Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones, Mol Microbiol, 55, 712, 10.1111/j.1365-2958.2004.04437.x Zeng, 2017, Rational design of an ultrasensitive quorum-sensing switch, ACS Synth Biol, 6, 1445, 10.1021/acssynbio.6b00367 Bassler, 1997, Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi, J Bacteriol, 179, 4043, 10.1128/jb.179.12.4043-4045.1997 Surette, 1999, Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production, Proc Natl Acad Sci USA, 96, 1639, 10.1073/pnas.96.4.1639 Pereira, 2013, AI-2-mediated signalling in bacteria, FEMS (Fed Eur Microbiol Soc) Microbiol Rev, 37, 156 Xue, 2009, LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing, Cell Res, 19, 1258, 10.1038/cr.2009.91 Wang, 2005, Cyclic AMP (cAMP) and cAMP receptor protein influence both synthesis and uptake of extracellular autoinducer 2 in Escherichia coli, J Bacteriol, 187, 2066, 10.1128/JB.187.6.2066-2076.2005 Ha, 2018, Evidence of link between quorum sensing and sugar metabolism in Escherichia coli revealed via cocrystal structures of LsrK and HPr, Sci. Adv., 4, eaar7063, 10.1126/sciadv.aar7063 Tsao, 2010, Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli, Metab Eng, 12, 291, 10.1016/j.ymben.2010.01.002 Hauk, 2016, Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression, Nucleic Acids Res, 44, 10515 Hauk, 2020, Homologous quorum sensing regulatory circuit: a dual-input genetic controller for modulating quorum sensing-mediated protein expression in E. coli, ACS Synth Biol, 9, 2692, 10.1021/acssynbio.0c00179 Dong, 2000, AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc Natl Acad Sci USA, 97, 3526, 10.1073/pnas.97.7.3526 Liu, 2008, Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures, Biochemistry, 47, 7706, 10.1021/bi800368y Fetzner, 2015, Quorum quenching enzymes, J Biotechnol, 201, 2, 10.1016/j.jbiotec.2014.09.001 Köse-Mutlu, 2019, Quorum quenching for effective control of biofouling in membrane bioreactor: a comprehensive review of approaches, applications, and challenges, Environ. Engineer. Res., 24, 543, 10.4491/eer.2018.380 Danino, 2010, A synchronized quorum of genetic clocks, Nature, 463, 326, 10.1038/nature08753 Kim, 2019, Long-range temporal coordination of gene expression in synthetic microbial consortia, Nat Chem Biol, 15, 1102, 10.1038/s41589-019-0372-9 Alnahhas, 2020, Majority sensing in synthetic microbial consortia, Nat Commun, 11, 1, 10.1038/s41467-020-17475-z Gu, 2020, Quorum sensing-based dual-function switch and its application in solving two key metabolic engineering problems, ACS Synth Biol, 9, 209, 10.1021/acssynbio.9b00290 Jiang, 2020, Two completely orthogonal quorum sensing systems with self-produced autoinducers enable automatic delayed cascade control, ACS Synth Biol, 9, 2588, 10.1021/acssynbio.0c00370 Duran-Nebreda, 2021, Synthetic lateral inhibition in periodic pattern forming microbial colonies, ACS Synth Biol, 10, 277, 10.1021/acssynbio.0c00318 Kobayashi, 2004, Programmable cells: interfacing natural and engineered gene networks, Proc Natl Acad Sci USA, 101, 8414, 10.1073/pnas.0402940101 McMillen, 2002, Synchronizing genetic relaxation oscillators by intercell signaling, Proc Natl Acad Sci Unit States Am, 99, 679, 10.1073/pnas.022642299 Prindle, 2012, A sensing array of radically coupled genetic ’biopixels', Nature, 481, 39, 10.1038/nature10722 Prindle, 2014, Rapid and tunable post-translational coupling of genetic circuits, Nature, 508, 387, 10.1038/nature13238 Baumgart, 2017, Synchronized DNA cycling across a bacterial population, Nat Genet, 49, 1282, 10.1038/ng.3915 Gonzalez-Flo, 2020, Two-component biosensors: unveiling the mechanisms of predictable tunability, ACS Synth Biol, 9, 1328, 10.1021/acssynbio.0c00010 Anesiadis, 2008, Dynamic metabolic engineering for increasing bioprocess productivity, Metab Eng, 10, 255, 10.1016/j.ymben.2008.06.004 Anesiadis, 2013, Analysis and design of a genetic circuit for dynamic metabolic engineering, ACS Synth Biol, 2, 442, 10.1021/sb300129j Soma, 2015, Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production, Metab Eng, 30, 7, 10.1016/j.ymben.2015.04.005 Gupta, 2017, Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit, Nat Biotechnol, 35, 273, 10.1038/nbt.3796 Kim, 2017, Autonomous control of metabolic state by a quorum sensing (QS)-mediated regulator for bisabolene production in engineered E. coli, Metab Eng, 44, 325, 10.1016/j.ymben.2017.11.004 Doong, 2018, Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli, Proc Natl Acad Sci Unit States Am, 115, 201716920, 10.1073/pnas.1716920115 Zargar, 2016, Enhancing intercellular coordination: rewiring quorum sensing networks for increased protein expression through autonomous induction, ACS Synth Biol, 5, 923, 10.1021/acssynbio.5b00261 Dinh, 2019, Development of an autonomous and bifunctional quorum-sensing circuit for metabolic flux control in engineered Escherichia coli, Proc Natl Acad Sci USA, 116, 25562, 10.1073/pnas.1911144116 Shen, 2019, Combining directed evolution of pathway enzymes and dynamic pathway regulation using a quorum-sensing circuit to improve the production of 4-hydroxyphenylacetic acid in Escherichia coli, Biotechnol Biofuels, 12, 1, 10.1186/s13068-019-1438-3 Bao, 2019, Quorum-sensing based small RNA regulation for dynamic and tuneable gene expression, Biotechnol Lett, 41, 1147, 10.1007/s10529-019-02719-w Dinh, 2020, Development of a quorum-sensing based circuit for control of coculture population composition in a naringenin production system, ACS Synth Biol, 9, 590, 10.1021/acssynbio.9b00451 Soma, 2021, Design of synthetic quorum sensing achieving induction timing- independent signal stabilization for dynamic metabolic engineering of E. coli, ACS Synth Biol, 10, 1384, 10.1021/acssynbio.1c00008 Lu, 2019, Modular metabolic engineering for biobased chemical production, Trends Biotechnol, 37, 152, 10.1016/j.tibtech.2018.07.003 Chae, 2017, Recent advances in systems metabolic engineering tools and strategies, Curr Opin Biotechnol, 47, 67, 10.1016/j.copbio.2017.06.007 Ceroni, 2018, Burden-driven feedback control of gene expression, Nat Methods, 15, 387, 10.1038/nmeth.4635 Xu, 2020, Pyruvate-responsive genetic circuits for dynamic control of central metabolism, Nat Chem Biol, 16, 1261, 10.1038/s41589-020-0637-3 Bittihn, 2020, Genetically engineered control of phenotypic structure in microbial colonies, Nature Microbiology, 5, 697, 10.1038/s41564-020-0686-0 McCarty, 2020, Multiplexed CRISPR technologies for gene editing and transcriptional regulation, Nat Commun, 11, 1, 10.1038/s41467-020-15053-x Santos-Moreno, 2020, CRISPR-based gene expression control for synthetic gene circuits, Biochem Soc Trans, 48, 1979, 10.1042/BST20200020 Reis, 2019, Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays, Nat Biotechnol, 37, 1294, 10.1038/s41587-019-0286-9 Wang, 2017, Mini photobioreactors for in vivo real-time characterization and evolutionary tuning of bacterial optogenetic circuit, ACS Synth Biol, 6, 1793, 10.1021/acssynbio.7b00091 Zhao, 2018, Optogenetic regulation of engineered cellular metabolism for microbial chemical production, Nature, 555, 683, 10.1038/nature26141 Carrasco-López, 2020, Optogenetics and biosensors set the stage for metabolic cybergenetics, Curr Opin Biotechnol, 65, 296, 10.1016/j.copbio.2020.07.012 Pouzet, 2020, The promise of optogenetics for bioproduction: dynamic control strategies and scale-up instruments, Bioengineering, 7, 1, 10.3390/bioengineering7040151 Steel, 2020, In situ characterisation and manipulation of biological systems with Chi.Bio, PLoS Biol, 18, 1, 10.1371/journal.pbio.3000794 Lalwani, 2021, Optogenetic control of the lac operon for bacterial chemical and protein production, Nat Chem Biol, 17, 71, 10.1038/s41589-020-0639-1 E. Romano, A. Baumschlager, E. B. Akmeriç, N. Palanisamy, M. Houmani, G. Schmidt, M. A. Öztürk, L. Ernst, M. Khammash, B. Di Ventura, Engineering AraC to make it responsive to light instead of arabinose, Nat Chem Biol:10.1038/s41589-021-00787-6. Tschirhart, 2017, Electronic control of gene expression and cell behaviour in Escherichia coli through redox signalling, Nat Commun, 8, 1, 10.1038/ncomms14030 Bhokisham, 2020, A redox-based electrogenetic CRISPR system to connect with and control biological information networks, Nat Commun, 11, 1, 10.1038/s41467-020-16249-x Vanarsdale, 2020, A coculture based tyrosine-tyrosinase electrochemical gene circuit for connecting cellular communication with electronic networks, ACS Synth Biol, 9, 1117, 10.1021/acssynbio.9b00469 Selberg, 2018, The potential for convergence between synthetic biology and bioelectronics, Cell Sys., 7, 231, 10.1016/j.cels.2018.08.007 Zhang, 2020, Living electronics, Nano Research, 13, 1205, 10.1007/s12274-019-2570-x Dixon, 2021, Sensing the future of bio-informational engineering, Nat Commun, 12, 1, 10.1038/s41467-020-20764-2 Basu, 2005, A synthetic multicellular system for programmed pattern formation, Nature, 434, 1130, 10.1038/nature03461 Tabor, 2009, A synthetic genetic edge detection program, Cell, 137, 1272, 10.1016/j.cell.2009.04.048 Liu, 2011, Sequential establishment of stripe patterns in an expanding cell population, Science, 334, 238, 10.1126/science.1209042 Kong, 2017, Engineering robust and tunable spatial structures with synthetic gene circuits, Nucleic Acids Res, 45, 1005, 10.1093/nar/gkw1045 Walker, 2019, Engineered cell-to-cell signalling within growing bacterial cellulose pellicles, Micriob. Biotechn., 12, 611, 10.1111/1751-7915.13340 Curatolo, 2020, Cooperative pattern formation in multi-component bacterial systems through reciprocal motility regulation, Nat Phys, 16, 1152, 10.1038/s41567-020-0964-z Brenner, 2007, Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium, Proc Natl Acad Sci USA, 104, 17300, 10.1073/pnas.0704256104 Tamsir, 2011, Robust multicellular computing using genetically encoded NOR gates and chemical ’wiresg’, Nature, 469, 212, 10.1038/nature09565 Regot, 2011, Distributed biological computation with multicellular engineered networks, Nature, 469, 207, 10.1038/nature09679 Wiesner, 2012, Lasp-1 regulates podosome function, PloS One, 7, 1 Hu, 2015, Programming the quorum sensing-based and gate in Shewanella oneidensis for logic gated-microbial fuel cells, Chem Commun, 51, 4184, 10.1039/C5CC00026B Urrios, 2018, Plug-and-Play multicellular circuits with time-dependent dynamic responses, ACS Synth Biol, 7, 1095, 10.1021/acssynbio.7b00463 Sardanyés, 2015, Computational implementation of a tunable multicellular memory circuit for engineered eukaryotic consortia, Front Physiol, 6, 281, 10.3389/fphys.2015.00281 Urrios, 2016, A synthetic multicellular memory device, ACS Synth Biol, 5, 862, 10.1021/acssynbio.5b00252 Macia, 2017, Synthetic associative learning in engineered multicellular consortia, J R Soc Interface, 14, 20170158, 10.1098/rsif.2017.0158 Chen, 2015, Emergent genetic oscillations in a synthetic microbial consortium, Science, 349, 986, 10.1126/science.aaa3794 You, 2004, Programmed population control by cell-cell communication and regulated killing, Nature, 428, 868, 10.1038/nature02491 Balagaddé, 2008, A synthetic Escherichia coli predator-prey ecosystem, Mol Syst Biol, 4, 1, 10.1038/msb.2008.24 Bittihn, 2018, Rational engineering of synthetic microbial systems : from single cells to consortia, Curr Opin Microbiol, 45, 92, 10.1016/j.mib.2018.02.009 Stephens, 2019, Bacterial co-culture with cell signaling translator and growth controller modules for autonomously regulated culture composition, Nat Commun, 10, 1, 10.1038/s41467-019-12027-6 McCardell, 2017, Control of bacterial population density with population feedback and molecular sequestration, bioRxiv, 225045 Fedorec, 2021, Single strain control of microbial consortia, Nat Commun, 12, 1, 10.1038/s41467-021-22240-x Liu, 2020, Dynamic cell programming with quorum sensing-controlled CRISPRi circuit, ACS Synth Biol, 9, 1284, 10.1021/acssynbio.0c00148 Miano, 2020, Inducible cell-to-cell signaling for tunable dynamics in microbial communities, Nat Commun, 11, 1, 10.1038/s41467-020-15056-8 Liao, 2019, Rock-paper-scissors: engineered population dynamics increase genetic stability, Science, 365, 1045, 10.1126/science.aaw0542 Karkaria, 2021, Automated design of synthetic microbial communities, Nat Commun, 12, 1, 10.1038/s41467-020-20756-2 Brockman, 2015, Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites, Metab Eng, 28, 104, 10.1016/j.ymben.2014.12.005 Ren, 2018, Population regulation in microbial consortia using dual feedback control, 5341 Fiore, 2017, In-silico analysis and implementation of a multicellular feedback control strategy in a synthetic bacterial consortium, ACS Synth Biol, 6, 507, 10.1021/acssynbio.6b00220 Fiore, 2021