Synthetic Biological Circuits within an Orthogonal Central Dogma
2020; Elsevier BV; Volume: 39; Issue: 1 Linguagem: Inglês
10.1016/j.tibtech.2020.05.013
ISSN0167-9430
AutoresAlan Costello, Ahmed H. Badran,
Tópico(s)CRISPR and Genetic Engineering
ResumoDevelopment of fully synthetic nucleobase pairs that faithfully interact in living cells, and their applications in creating semisynthetic organisms with expanded and orthogonal information-carrying capacity.Harnessing naturally occurring and mutually orthogonal DNA replication systems to enable replication of target genes. Highly error-prone variations on these systems enable robust directed evolution of biomolecules.Engineering and directed evolution of mutually orthogonal transcription factors that operate with high dynamic range, low background, and respond to a wide repertoire of stimuli in vivo.Recent developments in in vivo orthogonal protein translation including: orthogonal RBS–orthogonal anti-RBS pairs, covalently linked rRNA subunits to discover novel enzymatic capabilities, improved incorporation of non-canonical amino acids and decoding quadruplet codons. Synthetic biology strives to reliably control cellular behavior, typically in the form of user-designed interactions of biological components to produce a predetermined output. Engineered circuit components are frequently derived from natural sources and are therefore often hampered by inadvertent interactions with host machinery, most notably within the host central dogma. Reliable and predictable gene circuits require the targeted reduction or elimination of these undesirable interactions to mitigate negative consequences on host fitness and develop context-independent bioactivities. Here, we review recent advances in biological orthogonalization, namely the insulation of researcher-dictated bioactivities from host processes, with a focus on systematic developments that may culminate in the creation of an orthogonal central dogma and novel cellular functions. Synthetic biology strives to reliably control cellular behavior, typically in the form of user-designed interactions of biological components to produce a predetermined output. Engineered circuit components are frequently derived from natural sources and are therefore often hampered by inadvertent interactions with host machinery, most notably within the host central dogma. Reliable and predictable gene circuits require the targeted reduction or elimination of these undesirable interactions to mitigate negative consequences on host fitness and develop context-independent bioactivities. Here, we review recent advances in biological orthogonalization, namely the insulation of researcher-dictated bioactivities from host processes, with a focus on systematic developments that may culminate in the creation of an orthogonal central dogma and novel cellular functions. Natural gene circuits (see Glossary) rely on regulatory factors to govern cellular activities, leveraging elements of both circuit insulation and interaction to afford the necessary responses [1.Müller I.E. et al.Gene networks that compensate for crosstalk with crosstalk.Nat. Commun. 2019; 10: 1-8Crossref PubMed Scopus (3) Google Scholar]. These natural gene circuits are maintained through discrete functional modules, which allow for both robustness and plasticity in adapting to changing environmental conditions [2.Hartwell L.H. et al.From molecular to modular cell biology.Nature. 1999; 402: C47-C52Crossref PubMed Google Scholar]. Over the past two decades, components of natural gene circuits have been isolated and repurposed to develop increasingly complex engineered gene circuits [3.Cameron D.E. et al.A brief history of synthetic biology.Nat. Rev. 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Furthermore, empirical approaches for improving synthetic circuits and their components become particularly challenging when circuits grow in complexity, as the combinations of possible modifications grows exponentially [13.Gyorgy A. et al.Isocost lines describe the cellular economy of genetic circuits.Biophys. J. 2015; 109: 639-646Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. To date, bottlenecks in engineering more complex gene circuits have stemmed from two broad factors: (i) engineered circuits typically repurpose components that are optimized to function within their native contexts, and which are not immediately suited for user-defined objectives, such as high levels of basal expression in an off-state [20.Haseltine E.L. Arnold F.H. Synthetic gene circuits: design with directed evolution.Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 1-19Crossref PubMed Scopus (100) Google Scholar]; and (ii) their performance is often limited by incompatibility of components, such as crosstalk or toxicity in the chosen host [21.Ellefson J.W. et al.Directed evolution of genetic parts and circuits by compartmentalized partnered replication.Nat. Biotechnol. 2014; 32: 97-101Crossref PubMed Scopus (92) Google Scholar]. Insulation of these components from host processes (biological orthogonalization; Box 1) has been described as early as the 1960s [22.Jacob F. Monod J. Genetic regulatory mechanisms in the synthesis of proteins.J. Mol. Biol. 1961; 3: 318-356Crossref PubMed Google Scholar] (Figure 1, Key Figure) and has significantly improved the predictability of engineered circuits by limiting known instances of cellular crosstalk [5.Meyer A.J. et al.Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors.Nat. Chem. Biol. 2019; 15: 196-204Crossref PubMed Scopus (126) Google Scholar]. An orthogonal and user-controlled paralog [23.Liu C.C. et al.Toward an orthogonal central dogma.Nat. Chem. Biol. 2018; 14: 103-106Crossref PubMed Scopus (62) Google Scholar] of the central dogma may therefore continue to improve the reliability of engineered circuits, and enable increasingly numerous elements to coexist alongside host components without adverse cellular effects. In this review, we discuss prior studies aimed at segregating synthetic and host elements, and take inspiration from native biological systems to selectively partition, compartmentalize, or orthogonalize native and/or engineered components to enable specific goals. As modern engineered gene circuits continue to rely heavily on the host central dogma, we discuss these developments with a focus on their integration to furnish an orthogonal central dogma, wherein elements of information maintenance, transfer, and translation have been modified and/or insulated from host interactions.Box 1Biological OrthogonalizationThe term orthogonal or orthogonality in synthetic biology describes the inability of two or more biomolecules, similar in composition and/or function, to interact with one another or affect their respective substrates. For example, two proteases may be mutually orthogonal if they are unable to cleave one another's respective substrates, or two aaRSs that do not cross aminoacylate their noncognate tRNA. By necessity, all biomolecules within a cell should not interact directly with one another. This allows key processes such as those within the central dogma to perform discrete functions (replication, transcription, and translation) while remaining intrinsically linked as each function is dependent on the prior stages. As it relates to the development of engineered gene circuits, the necessary degree of orthogonality depends on the user-defined objectives. For example, it may be necessary to orthogonally control the transcription of multiple genes using independent promoters in an engineered gene circuit. Here, TFs with known mutual orthogonality in their respective operator sequences may be sufficient. Conversely, more complex goals such as the large-scale implementation of expanded genetic codes will undoubtedly require a larger repertoire of orthogonal elements. The term orthogonal or orthogonality in synthetic biology describes the inability of two or more biomolecules, similar in composition and/or function, to interact with one another or affect their respective substrates. For example, two proteases may be mutually orthogonal if they are unable to cleave one another's respective substrates, or two aaRSs that do not cross aminoacylate their noncognate tRNA. By necessity, all biomolecules within a cell should not interact directly with one another. This allows key processes such as those within the central dogma to perform discrete functions (replication, transcription, and translation) while remaining intrinsically linked as each function is dependent on the prior stages. As it relates to the development of engineered gene circuits, the necessary degree of orthogonality depends on the user-defined objectives. For example, it may be necessary to orthogonally control the transcription of multiple genes using independent promoters in an engineered gene circuit. Here, TFs with known mutual orthogonality in their respective operator sequences may be sufficient. Conversely, more complex goals such as the large-scale implementation of expanded genetic codes will undoubtedly require a larger repertoire of orthogonal elements. Engineered gene circuits can be introduced into a host chassis as stand-alone episomes [24.Kittleson J.T. et al.Rapid optimization of gene dosage in E. coli using DIAL strains.J. Biol. Eng. 2011; 5: 10Crossref PubMed Scopus (0) Google Scholar] or integrated into the host genome [25.Gaidukov L. et al.A multi-landing pad DNA integration platform for mammalian cell engineering.Nucleic Acids Res. 2018; 46: 4072-4086Crossref PubMed Scopus (48) Google Scholar]. As inadvertent recognition of engineered sequences by host components may affect circuit performance in unpredictable ways, mechanisms that insulate exogenous genetic information from host machinery would limit reliance on the host for replication or gene expression [26.Weigele P. Raleigh E.A. Biosynthesis and function of modified bases in bacteria and their viruses.Chem. Rev. 2016; 116: 12655-12687Crossref PubMed Scopus (72) Google Scholar]. Epigenetic modification of nucleic acids is among the most common mechanisms exploited by natural biological systems to insulate specific cellular processes. Taking inspiration from these natural systems, Khalil and colleagues recently established an orthogonal methodology to control gene transcription in eukaryotic cells through epigenetic signaling [27.Park M. et al.Engineering epigenetic regulation using synthetic read-write modules.Cell. 2019; 176: 227-238.e20Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar]. This system uses a modified nucleobase commonly found in prokaryotes but absent in eukaryotic genomes, N6-methyldeoxyadenosine (m6dA), by porting the requisite methyltransferase and transcription factors to enable efficient and orthogonal information storage and propagation (Figure 2A ). A diversity of DNA modifications exists in natural genomes [28.Sood A.J. et al.DNAmod: the DNA modification database.J. Cheminform. 2019; 11: 30Crossref PubMed Scopus (6) Google Scholar] (Figure 2B), wherein the abundance of non-canonical nucleobases may make their genetic information innately orthogonal to the host machinery. For example, the genomes of a eukaryotic multicellular organism may be identical in sequence but produce distinct gene expression patterns through alternative epigenetic profiles [27.Park M. et al.Engineering epigenetic regulation using synthetic read-write modules.Cell. 2019; 176: 227-238.e20Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar]. Such modifications may also provide protective functions to cellular circuits. Bacteriophage genomes can have partial or complete substitution of nucleobases by modified variants [26.Weigele P. Raleigh E.A. Biosynthesis and function of modified bases in bacteria and their viruses.Chem. Rev. 2016; 116: 12655-12687Crossref PubMed Scopus (72) Google Scholar], which are thought to resist endonuclease-mediated digestion upon infection. However, incorporation of non-canonical nucleobases may require the action of dedicated polymerases to facilitate replication and propagation [29.Warren R.A.J. Modified bases in bacteriophage DNAs.Annu. Rev. Microbiol. 1980; 34: 137-158Crossref PubMed Google Scholar]. Modified nucleotides in bacteriophages are generated primarily through enzymatic modification of cellular precursors [26.Weigele P. Raleigh E.A. Biosynthesis and function of modified bases in bacteria and their viruses.Chem. Rev. 2016; 116: 12655-12687Crossref PubMed Scopus (72) Google Scholar]; however, some bacteriophage components can introduce secondary nucleotide modifications following replication [26.Weigele P. Raleigh E.A. Biosynthesis and function of modified bases in bacteria and their viruses.Chem. Rev. 2016; 116: 12655-12687Crossref PubMed Scopus (72) Google Scholar,30.Gommers-Ampt J.H. Borst P. Hypermodified bases in DNA.FASEB J. 1995; 9: 1034-1042Crossref PubMed Google Scholar]. It may therefore be possible to exploit these modifications to program additional layers of orthogonal information maintenance in non-natural hosts. The observation of non-canonical nucleobases in natural systems has motivated significant interest in incorporating synthetic nucleotide pairs into cellular DNA, where such efforts could enable genetic code expansion through increased sequence diversity. Recent work to expand the genetic code has recently resulted in accretion from the canonical four to six [31.Yang Z. et al.Amplification, mutation, and sequencing of a six-letter synthetic genetic system.J. Am. Chem. Soc. 2011; 133: 15105-15112Crossref PubMed Scopus (159) Google Scholar, 32.Dhami K. et al.Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet.Nucleic Acids Res. 2014; 42: 10235-10244Crossref PubMed Scopus (51) Google Scholar, 33.Hirao I. Kimoto M. Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma.Proc. Jpn. Acad. Ser. B Phys. Biol. 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Finally, nucleic acid phosphate backbones are known to be extensively modified to phosphorothioate across diverse bacterial genera [37.Wang L. et al.DNA phosphorothioation is widespread and quantized in bacterial genomes.PNAS. 2011; 108: 2963-2968Crossref PubMed Scopus (0) Google Scholar]; an interesting example of an artificial innovation that was later discovered to occur naturally in biological systems. Recent work by Holliger and colleagues extended this observation by creating alkyl phosphonate nucleic acids, showcasing how such modifications can give rise to novel biomolecular interactions in evolved aptamers [38.Arangundy-Franklin S. et al.A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids.Nat. Chem. 2019; 11: 533-542Crossref PubMed Scopus (10) Google Scholar]. Although non-canonical nucleobases may limit interactions with host machinery, a complementary mechanism leverages orthogonal replication of genetic information. Among well-studied systems, orthogonal replication of the lytic Bacillus ϕ29 bacteriophage genome is perhaps the most well understood, and used as a model for protein-primed replication [39.Salas M. et al.DNA-binding proteins essential for protein-primed bacteriophage ϕ29 DNA replication.Front. Mol. Biosci. 2016; 3: 37Crossref PubMed Google Scholar]. ϕ29 DNA replication is initiated asynchronously at both ends of the linear genome, wherein a terminal protein (TP)–DNA polymerase (DNAP) heterodimer recognizes the protein-capped replication origins (Figure 2C). The DNAP then dissociates from the TP and continues processive elongation coupled to strand displacement. Continuous elongation by two DNAPs gives rise to the complete duplication of the parental strands [39.Salas M. et al.DNA-binding proteins essential for protein-primed bacteriophage ϕ29 DNA replication.Front. Mol. Biosci. 2016; 3: 37Crossref PubMed Google Scholar]. Taking inspiration from natural viral replication systems, ϕ29 bacteriophage components have enabled minimally self-replicating DNA systems in vitro (Figure 2D). Ichihashi and colleagues developed a minimal transcription- and translation-coupled DNA replication (TTcDR) system wherein a plasmid encoding its own DNAP replicates by rolling circle amplification [40.Sakatani Y. et al.Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination.Sci. Rep. 2018; 8: 1-11Crossref PubMed Scopus (11) Google Scholar]. Following amplification, Cre recombinase loxP sites facilitate recircularization of the nascent DNA, with rounds of directed evolution used to improve circularization efficiency by 60-fold. Danelon and colleagues [7.van Nies P. et al.Self-replication of DNA by its encoded proteins in liposome-based synthetic cells.Nat. 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A minimal mitochondrial DNA replication system has been recapitulated in vitro, consisting of only of two protein factors: DNA polymerase POLγ and TWINKLE helicase [41.Korhonen J.A. et al.Reconstitution of a minimal mtDNA replisome in vitro.EMBO J. 2004; 23: 2423-2429Crossref PubMed Scopus (265) Google Scholar]. This system has not yet been extended to a heterologous host, likely a reflection of slow polymerase elongation rate (280 bp/min) and a requirement for two asymmetric origins of replication. Recently, an orthogonal DNA replication (OrthoRep) system has been established in yeast to realize cellular mutation rates beyond the error catastrophe threshold while mitigating negative consequences on host fitness (Figure 2E) [42.Ravikumar A. et al.An orthogonal DNA replication system in yeast.Nat. Chem. Biol. 2014; 10: 175-177Crossref PubMed Scopus (55) Google Scholar, 43.Arzumanyan G.A. et al.Mutually orthogonal DNA replication systems in vivo.ACS Synth. 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Beyond the development and maintenance of orthogonal DNA, mechanisms for insulating transcriptional and regulatory elements have historically served as the foundation for synthetically controlling gene expression, building upon pioneering studies of inducible systems [47.de Boer H.A. et al.The tac promoter: a functional hybrid derived from the trp and lac promoters.Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 21-25Crossref PubMed Google Scholar, 48.Skerra A. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli.Gene. 1994; 151: 131-135Crossref PubMed Google Scholar, 49.Guzman L.M. et al.Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3781) Google Scholar]. Rules governing model inducer-responsive transcriptional units have been established and natural regulatory components have been exploited through engineering and directed evolution to yield synthetic paralogs more suited to their new contexts [5.Meyer A.J. et al.Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors.Nat. Chem. Biol. 2019; 15: 196-204Crossref PubMed Scopus (126) Google Scholar]. Inducible systems classically consist of three components: a protein transcription factor (TF); a DNA motif specifically bound by the TF; and a nontoxic, cell-permeable small molecule that promotes or inhibits the binding of a TF to its cognate DNA motif (Figure 3A ). Although not typically identified as orthogonal systems during their development, the success of small molecule-inducible systems has derived from significant efforts to insulate their functionality from host machinery and to limit crosstalk with pre-existing components [5.Meyer A.J. et al.Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors.Nat. Chem. Biol. 2019; 15: 196-204Crossref PubMed Scopus (126) Google Scholar]. This includes the development of nonmetabolizable (e.g., isopropyl β-d-1-thiogalactopyranoside; IPTG) or nontoxic (e.g., anhydrotetracycline; aTc) inducers, elimination of native pathways for inducer detection or catabolism (e.g., lactose and arabinose catabolism), or modulation of TFs to mitigate pleiotropic effects (e.g., IPTG and arabinose crosstalk) [50.Lee S.K. et al.Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters.Appl. Environ. 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