- Email: [email protected]
Principles of Systems Biology, No. 14
Cell Systems
Cell Systems Call Principles of Systems Biology, No. 14 This month: sage advice from phage to their offspring; systematic analyses of protein quality control, mitochondrial respiration, and woody biomass; a continental-scale experiment; and engineered protein tools galore. Peptide-Based Communication in Phages
Molecular Decision-Making of Protein Fate
Links in the Mitochondrial Interactome
Zohar Erez and Rotem Sorek, Weizmann Institute of Science
Sichen Shao, Harvard Medical School; Ramanujan S. Hegde, MRC Laboratory of Molecular Biology
Devin K. Schweppe, Juan D. Chavez, and James E. Bruce, University of Washington
Principles
Mitochondria produce energy for living cells, and their function is mediated through protein conformations and interaction networks. In Schweppe and Chavez et al. (Schweppe et al., Proc. Natl. Acad. Sci. USA, published online January 27, 2017. https://dx.doi.org/ 10.1073/pnas.1617220114), we apply protein interaction reporter (PIR) technologies in a systems-level study of molecular interactions inside intact, respiring mitochondria. These efforts reveal a network comprised of 2,049 cross-linked peptide pairs from more than one-third of the mitochondrial proteome. Interactions were observed between proteins throughout the electron transport chain complexes, ATP synthase, and the MICOS complex. Cross-linked sites provide physical constraints for protein docking to visualize previously unresolved complex subunits. PIR data also established direct physical evidence of the existence of the Complex I-III ‘‘respirasome’’ in respiring mitochondria, enabling prediction of interfacial regions among supercomplex assemblies.
Principles A temperate phage is a phage that can choose between the lytic and lysogenic cycles. In the lytic cycle, that phage replicates within its host bacterial cell and eventually kills it, while in the lysogenic cycle the phage genome integrates into the bacterial chromosome and the phage becomes dormant. We have recently discovered that phages infecting Bacillus subtilis use a small-molecule communication system to coordinate the lysis-lysogeny decision (Erez et al., Nature 541, 488–493). When the phage first encounters a bacterial colony, it preferentially chooses the lytic cycle. During infection, the phage produces a short communication peptide that is released into the medium. Later generations of the phage measure the amount of the communication peptide and choose the lysogenic cycle if the amount is sufficiently high. This peptide therefore acts as a signaling molecule through which earlier generation of the phage notify the later generations how many successful infections have recently occurred.
.earlier generation of the phage notify the later generations how many successful infections have recently occurred. What’s Next? So far, we found the phage communication system mostly in one family of phages infecting Bacillus subtilis. We are now looking into whether such communication systems are more widespread in nature.
Cells make millions of new proteins every minute. Each nascent protein usually engages cellular factors, such as chaperones, to facilitate its correct maturation. If maturation fails, quality control factors must route the protein for degradation. How a mixture of maturation and quality control factors collude to make these critical decisions about a nascent protein’s fate is not understood. We biochemically rebuilt a protein triage system with purified components to decipher how fate decisions are made (Shao et al., Science 355, 298–302). Using time-resolved photocrosslinking, we followed the flux of a radiolabeled substrate through a triage system composed of three interacting chaperones. The most promiscuous and rapidly binding chaperone captures the substrate first, but can only retain it for 30 s. This places a time limit for completing a private and rapid substrate transfer to the second chaperone, which is dedicated to protein biosynthesis. If time runs out before this transfer, substrates released from the first chaperone are engaged by the third chaperone, which is dedicated to protein degradation. This explains how biosynthesis is prioritized over degradation, but only for a limited time.
We biochemically rebuilt a protein triage system with purified components to decipher how fate decisions are made. What’s Next? It will be important to understand in atomic detail how private transfer reactions between chaperones work, the extent to which principles from this work apply to other multichaperone systems of protein quality control, and the roles inappropriate triage play in various diseases of protein misfolding.
140 Cell Systems 4, February 22, 2017 ª 2017 Published by Elsevier Inc.
Principles
.a network comprised of 2,049 cross-linked peptide pairs from more than a third of the mitochondrial proteome. What’s Next? Each identified link now serves as a molecular probe, allowing us, or virtually any lab, to perform quantification of conformationdependent protein interfaces and dynamic protein complex assembly. Comparative network analysis of normal versus disease, young versus old, or mitochondria from different tissues will significantly advance understanding of mitochondrial function and the molecular basis of pathological phenotypes.
Cell Systems
Cell Systems Call Network Analysis of Woody Biomass
Do Fungi Regulate Forest Dynamics?
Eshchar Mizrachi, Lieven Verbeke, Yves Van de Peer, Kathleen Marchal, and Alexander A. Myburg, University of Pretoria, Ghent University
Jonathan A. Bennett and John Klironomos, University of British Columbia - Okanagan
Principles
Woody biomass from trees accounts for a significant amount of the world’s sequestered carbon and is of high value as a renewable feedstock for energy and biomaterials. However, traits associated with its beneficiation are complex, i.e., impacted by hundreds of interacting genes, as well as environmental cues, over the long lifetime of a tree. Systems genetics, or the combined genotyping and molecular trait profiling of segregating individuals, offers vast potential to understand the molecular mechanisms affecting complex plant traits. We developed network-based data integration (Mizrachi et al., Proc. Natl. Acad. Sci. USA, published online January 17, 2017. https://dx.doi.org/10.1073/pnas. 1620119114), a method in which genotyping data, expression profiles, gene expression polymorphisms (eQTLs), and prior information on pathways and biological functions were simultaneously used to prioritize genes, loci, and pathways associated with growth, biomass-accumulation, and processing traits in Eucalyptus.
Every plant interacts with a multitude of organisms in the soil, which can have dramatic effects on the survival and growth of their offspring. These interactions, termed plant-soil feedbacks, vary from positive to negative and are hypothesized as a major driver of biodiversity. However, the factors determining plant-soil feedbacks are only partially understood. Using a continental-scale experiment that included 55 species and 550 populations of temperate trees, we showed that the type of mycorrhizal fungi associated with the roots of the tree was a strong predictor of their plant-soil feedback (Bennett et al., Science 355, 181–184). Seedlings of trees with ectomycorrhizal fungi responded positively to soil collected beneath adult trees, whereas seedlings of trees with arbuscular mycorrhizal fungi responded negatively, largely owing to differences in their ability to protect the seedlings from pathogens and other antagonists. Importantly, these effects could be linked to tree population dynamics at both local and national scales, highlighting their importance for regulating forest dynamics.
Systems genetics. offers vast potential to understand the molecular mechanisms affecting complex plant traits.
.the type of mycorrhizal fungi associated with the roots of the tree was a strong predictor of their plant-soil feedback.
Principles
What’s Next?
What’s Next?
We propose pathways for biotechnological engineering or molecular breeding of lignocellulosic biomass properties. Such intervention at these pathways should be tested empirically, and natural variation in these pathways should be explored for predictive breeding. The developed method, NBDI, is highly applicable to any scenario for which systems genetics data are available. We envision that NBDI, but also other networkbased methods, will become key to the holistic interpretation and evolutionary understanding of trait variation by bridging the gap between forward genetics and systems biology.
Mycorrhizal fungi are just one factor determining plant-soil feedback and population dynamics. Incorporating other characteristics of the tree species and the environment in which the tree is found should improve our understanding of plant-soil feedback and forest dynamics in general.
Electronically Controlling Biology: Exploiting Nature’s Electron Carriers Gregory F. Payne and William E. Bentley, University of Maryland, College Park
Principles For years, we have been observing and altering biological functions through the voltages and currents associated with the movement of ions (e.g., neural stimulators, defibrillators, etc.). Biological functions— including their communication networks— are also governed by the flow of electrons through oxidation and reduction reactions. We recently demonstrated that by transferring electrons through such redox molecules (nature’s ‘‘electron’’ carriers), we can actuate gene expression in Escherichia coli (Tschirhart et al., Nat. Comm., published online January 17, 2017, https://dx.doi.org/ 10.1038/ncomms14030). Specifically, we used electrodes to oxidize pyocyanin, a redox active biological signal molecule that activates soxS-promoted gene expression in E. coli. In cells with an attenuated oxidative stress response regulon, we demonstrated a linear increase in gene expression with total electrical charge. By reversing the applied potential, we electronically turned ‘‘off’’ gene expression. Using this electrogenetic switch, we actuated several biological phenotypes, including motility and quorum sensing activity in nearby cells.
.we demonstrated a linear increase in gene expression with total electrical charge. What’s Next? This opens the door to a variety of functions, perhaps even connecting consumer electronics to biological systems that interact with humans. We envision devices that incorporate bacteria, perhaps even those engineered to make human pharmaceuticals.
Cell Systems 4, February 22, 2017 141
Cell Systems
Cell Systems Call Virtual Human Gut Microbiota Ronan Fleming and Ines Thiele, University of Luxembourg.
Principles The human gut microbiota is a metabolically active community of 10–100 trillion organisms comprising 500–1,000 species. The gut microbiota contributes to essential functions for the human host, such as food digestion, synthesis of essential amino acids and vitamins, protection against pathogens, and maturation of the host immune system. Changes in human gut microbial composition have been associated with many chronic diseases. So far, microbiome studies have provided valuable insights into which microbes live in our gut and where they are located in the gastrointestinal tract. A next challenge is to elucidate how these microbes modulate host metabolism, thereby moving beyond association to establish causation. To complement existing in vitro and in vivo tools, we generated in silico genome-scale metabolic reconstructions for 773 human gut bacteria (Magnu´sdo´ttir et. al., Nat. Biotechnol. 35, 81–89). The corresponding constraint-based computational microbial models serve as a molecular mechanistic framework for integration with, for example, personalized metagenomic data, to infer the metabolic capacity of the unique microbiota of each individual.
.we generated in silico genome-scale metabolic reconstructions for 773 human gut bacteria. What’s Next? Combining individualized microbiota models with a comprehensive molecular systems physiology model of human metabolism would enable the generation of mechanistic hypotheses on how microbial metabolic activity modulates host metabolism. Furthermore, the integration of personalized dietary information with such computational models would permit personalized exploration of the diet-gut-health axis and help to accelerate biomedicine by disentangling confounding factors.
142 Cell Systems 4, February 22, 2017
De Novo Design of Mammalian Promoters Joseph K. Cheng, University of Texas at Austin and Seattle Children’s Research Institute; and Hal S. Alper, The University of Texas at Austin
Principles Traditional mammalian promoter engineering emphasized cis-elements or DNA motifs (e.g., transcription factor binding sites, TFBSs) in regions 50 of the core promoter. Many of these regions were identified by dissecting key viral or endogenous genes, but do not give immediate clues of how to build synthetic promoters. Recently, we demonstrated a generalizable workflow toward the design of synthetic mammalian promoters built from endogenous transcription factors (Cheng and Alper, ACS Synth. Biol., 5, 1455–1465). Our premise was straightforward: genes that are well expressed in a desired condition have promoters that are enriched with TFBSs that enable expression in that state. Ultimately, we hypothesized that TFBSs enriched in strong endogenous promoters make good synthetic parts for creating strong synthetic promoters. We demonstrate that establishing synthetic promoters constructed with these TFBSs did, in fact, establish strong endogenous promoters that were comparable to commonly referenced viral- and endogenous-derived promoters (hCMV-IE and EEF1A1 gene, respectively).
.TFBSs enriched in strong endogenous promoters make good synthetic parts for creating strong synthetic promoters. What’s Next? This general workflow relies on existing technologies (expression profiling and DNA synthesis), thus enabling its usage in diverse cell types. The ability to precisely control gene expression is not only crucial for therapeutic applications such as immunotherapy, but also paves the way to engineer the metabolism of mammalian cell types, expanding the utility of these cells to be more than just protein factories.
Probing RAS Signaling with a Computationally Engineered Rheostat John Rose and Dustin Maly, University of Washington
Principles Engineered proteins that are temporally regulated by chemical genetic or optogenetic inputs are powerful tools for studying complex, dynamic cellular processes. These synthetic protein systems allow researchers to precisely manipulate specific nodes with high temporal resolution, absent the pleiotropic effects of natural stimuli. To date, the majority of these engineered systems rely on multi-component intermolecular regulation. Single-component, intramolecular regulatory systems are more attractive for many applications, but their development has been hampered by the difficulty that is inherent in engineering allostery. In Rose et al. (Nat. Chem, Biol. 13, 119– 126), we devised a framework for computationally guided design of intramolecular regulatory systems, which enabled the development of a RAS rheostat, chemically inducible activator of RAS (CIAR). CIAR rapidly and tunably activates RAS upon addition of a small molecule. Using CIAR, we examined the wiring of the RAS/ERKsignaling network. We found that RAS/ ERK-signaling kinetics differ between cell lines and that the RAF inhibitor vemurafenib amplifies the magnitude of this signal. Global phosphoproteomics further classified nodes based on their dynamic responses to direct RAS activation via CIAR or EGF stimulation. The generality of our computational approach was further demonstrated by the design of two intramolecularly regulated Rho-family guanine exchange factors.
.computationally guided design of intramolecular regulatory systems.enabled the development of a RAS rheostat. What’s Next? CIAR will enable quantitative interrogation of dynamic RAS-signaling networks in a wide array of model systems. Furthermore, we envision our computationally guided framework will expand researchers’ abilities to develop intramolecular regulatory systems for precisely controlling complex biological processes with novel engineered protein tools.
Cell Systems
Cell Systems Call Tunable Thermal Bioswitches for In Vivo Control of Bacterial Therapeutics Dan Piraner, Mohamad Abedi, and Mikhail Shapiro, California Institute of technology
Principles While our ability to engineer complex genetic circuits that orchestrate the behavior of therapeutic microbes has rapidly advanced, we still lack tools allowing us to communicate with microbial agents after their deployment inside host organisms, for example, to deliver instructions based on the agents’ location in the body. To provide such capabilities, we engineered tunable transcriptional bioswitches that enable noninvasive remote control over cell function deep inside living organisms (Piraner et al., Nat. Chem. Biol. 13, 75–80). We demonstrated the utility of these sensors in three in vivo bacterial therapy applications, in which bacteria were activated with spatiotemporal precision via focused ultrasound, modulated their gene expression in response to fever, and triggered cell death upon leaving the body.
.tunable transcriptional bioswitches that enable noninvasive control over cell function deep inside living organisms. What’s Next? In this work, we introduced a new expandable toolbox that allows for highly spatiotemporally restricted control of cell function, and we demonstrated the ability to engineer temperature-sensitive genetic circuits. These temperature sensors can be used in applications ranging from bacterial therapy to controlling protein expression in bioreactors. In addition, these sensors could be systematically transferred to other organisms where they can extend the capabilities of existing techniques for genetic control, such as optically or chemically triggered gene expression, into deep-tissue in vivo applications.
Sense Codon Reassignment Boosts Protein Chemistries Zhenling Cui, Sergey Mureev, and Kirill Alexandrov, University of Queensland
Principles Incorporation of unnatural amino acids (uAAs) via reassigned codons is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. So far, the site-selective incorporation of multiple uAAs into polypeptides was hampered by the limited number of available nonsense codons. We recently described a method for preparing an Escherichia coli in vitro translation system depleted of specific endogenous tRNA isoacceptors corresponding to split codon boxes. Such a system is dependent on the addition of synthetic tRNA(s) for the respective sense codon(s) for the restoration of its translational activity (Cui et al., ACS Synth Biol., published online December 14, 2016. https://dx.doi.org/10. 1021/acssynbio.6b00245). This allows multisite-selective incorporation of unnatural amino acids, and we successfully applied this approach to produce the calmodulin protein harboring FRET-forming fluorescent probes.
.in vitro translation system depleted of specific endogenous tRNAs.allows introduction of multiple unnatural amino acids into the predefined positions. What’s Next? The successful reassignment of AGG sense codon to uAAs using our newly developed technique demonstrates the feasibility of creating a potentially large number of reassignable orthogonal codons in an E. coli cell-free translation system. This potentially allows a significant expansion of the codons available for reassignment and allows introduction of multiple unnatural amino acids into the predefined positions. Furthermore, this approach can be transferred to eukaryotic cell-free systems, where the introduction of uAAs is lagging behind that of E. coli systems.
Cell Systems 4, February 22, 2017 143
Cell Systems Call Principles of Systems Biology, No. 14 This month: sage advice from phage to their offspring; systematic analyses of protein quality control, mitochondrial respiration, and woody biomass; a continental-scale experiment; and engineered protein tools galore. Peptide-Based Communication in Phages
Molecular Decision-Making of Protein Fate
Links in the Mitochondrial Interactome
Zohar Erez and Rotem Sorek, Weizmann Institute of Science
Sichen Shao, Harvard Medical School; Ramanujan S. Hegde, MRC Laboratory of Molecular Biology
Devin K. Schweppe, Juan D. Chavez, and James E. Bruce, University of Washington
Principles
Mitochondria produce energy for living cells, and their function is mediated through protein conformations and interaction networks. In Schweppe and Chavez et al. (Schweppe et al., Proc. Natl. Acad. Sci. USA, published online January 27, 2017. https://dx.doi.org/ 10.1073/pnas.1617220114), we apply protein interaction reporter (PIR) technologies in a systems-level study of molecular interactions inside intact, respiring mitochondria. These efforts reveal a network comprised of 2,049 cross-linked peptide pairs from more than one-third of the mitochondrial proteome. Interactions were observed between proteins throughout the electron transport chain complexes, ATP synthase, and the MICOS complex. Cross-linked sites provide physical constraints for protein docking to visualize previously unresolved complex subunits. PIR data also established direct physical evidence of the existence of the Complex I-III ‘‘respirasome’’ in respiring mitochondria, enabling prediction of interfacial regions among supercomplex assemblies.
Principles A temperate phage is a phage that can choose between the lytic and lysogenic cycles. In the lytic cycle, that phage replicates within its host bacterial cell and eventually kills it, while in the lysogenic cycle the phage genome integrates into the bacterial chromosome and the phage becomes dormant. We have recently discovered that phages infecting Bacillus subtilis use a small-molecule communication system to coordinate the lysis-lysogeny decision (Erez et al., Nature 541, 488–493). When the phage first encounters a bacterial colony, it preferentially chooses the lytic cycle. During infection, the phage produces a short communication peptide that is released into the medium. Later generations of the phage measure the amount of the communication peptide and choose the lysogenic cycle if the amount is sufficiently high. This peptide therefore acts as a signaling molecule through which earlier generation of the phage notify the later generations how many successful infections have recently occurred.
.earlier generation of the phage notify the later generations how many successful infections have recently occurred. What’s Next? So far, we found the phage communication system mostly in one family of phages infecting Bacillus subtilis. We are now looking into whether such communication systems are more widespread in nature.
Cells make millions of new proteins every minute. Each nascent protein usually engages cellular factors, such as chaperones, to facilitate its correct maturation. If maturation fails, quality control factors must route the protein for degradation. How a mixture of maturation and quality control factors collude to make these critical decisions about a nascent protein’s fate is not understood. We biochemically rebuilt a protein triage system with purified components to decipher how fate decisions are made (Shao et al., Science 355, 298–302). Using time-resolved photocrosslinking, we followed the flux of a radiolabeled substrate through a triage system composed of three interacting chaperones. The most promiscuous and rapidly binding chaperone captures the substrate first, but can only retain it for 30 s. This places a time limit for completing a private and rapid substrate transfer to the second chaperone, which is dedicated to protein biosynthesis. If time runs out before this transfer, substrates released from the first chaperone are engaged by the third chaperone, which is dedicated to protein degradation. This explains how biosynthesis is prioritized over degradation, but only for a limited time.
We biochemically rebuilt a protein triage system with purified components to decipher how fate decisions are made. What’s Next? It will be important to understand in atomic detail how private transfer reactions between chaperones work, the extent to which principles from this work apply to other multichaperone systems of protein quality control, and the roles inappropriate triage play in various diseases of protein misfolding.
140 Cell Systems 4, February 22, 2017 ª 2017 Published by Elsevier Inc.
Principles
.a network comprised of 2,049 cross-linked peptide pairs from more than a third of the mitochondrial proteome. What’s Next? Each identified link now serves as a molecular probe, allowing us, or virtually any lab, to perform quantification of conformationdependent protein interfaces and dynamic protein complex assembly. Comparative network analysis of normal versus disease, young versus old, or mitochondria from different tissues will significantly advance understanding of mitochondrial function and the molecular basis of pathological phenotypes.
Cell Systems
Cell Systems Call Network Analysis of Woody Biomass
Do Fungi Regulate Forest Dynamics?
Eshchar Mizrachi, Lieven Verbeke, Yves Van de Peer, Kathleen Marchal, and Alexander A. Myburg, University of Pretoria, Ghent University
Jonathan A. Bennett and John Klironomos, University of British Columbia - Okanagan
Principles
Woody biomass from trees accounts for a significant amount of the world’s sequestered carbon and is of high value as a renewable feedstock for energy and biomaterials. However, traits associated with its beneficiation are complex, i.e., impacted by hundreds of interacting genes, as well as environmental cues, over the long lifetime of a tree. Systems genetics, or the combined genotyping and molecular trait profiling of segregating individuals, offers vast potential to understand the molecular mechanisms affecting complex plant traits. We developed network-based data integration (Mizrachi et al., Proc. Natl. Acad. Sci. USA, published online January 17, 2017. https://dx.doi.org/10.1073/pnas. 1620119114), a method in which genotyping data, expression profiles, gene expression polymorphisms (eQTLs), and prior information on pathways and biological functions were simultaneously used to prioritize genes, loci, and pathways associated with growth, biomass-accumulation, and processing traits in Eucalyptus.
Every plant interacts with a multitude of organisms in the soil, which can have dramatic effects on the survival and growth of their offspring. These interactions, termed plant-soil feedbacks, vary from positive to negative and are hypothesized as a major driver of biodiversity. However, the factors determining plant-soil feedbacks are only partially understood. Using a continental-scale experiment that included 55 species and 550 populations of temperate trees, we showed that the type of mycorrhizal fungi associated with the roots of the tree was a strong predictor of their plant-soil feedback (Bennett et al., Science 355, 181–184). Seedlings of trees with ectomycorrhizal fungi responded positively to soil collected beneath adult trees, whereas seedlings of trees with arbuscular mycorrhizal fungi responded negatively, largely owing to differences in their ability to protect the seedlings from pathogens and other antagonists. Importantly, these effects could be linked to tree population dynamics at both local and national scales, highlighting their importance for regulating forest dynamics.
Systems genetics. offers vast potential to understand the molecular mechanisms affecting complex plant traits.
.the type of mycorrhizal fungi associated with the roots of the tree was a strong predictor of their plant-soil feedback.
Principles
What’s Next?
What’s Next?
We propose pathways for biotechnological engineering or molecular breeding of lignocellulosic biomass properties. Such intervention at these pathways should be tested empirically, and natural variation in these pathways should be explored for predictive breeding. The developed method, NBDI, is highly applicable to any scenario for which systems genetics data are available. We envision that NBDI, but also other networkbased methods, will become key to the holistic interpretation and evolutionary understanding of trait variation by bridging the gap between forward genetics and systems biology.
Mycorrhizal fungi are just one factor determining plant-soil feedback and population dynamics. Incorporating other characteristics of the tree species and the environment in which the tree is found should improve our understanding of plant-soil feedback and forest dynamics in general.
Electronically Controlling Biology: Exploiting Nature’s Electron Carriers Gregory F. Payne and William E. Bentley, University of Maryland, College Park
Principles For years, we have been observing and altering biological functions through the voltages and currents associated with the movement of ions (e.g., neural stimulators, defibrillators, etc.). Biological functions— including their communication networks— are also governed by the flow of electrons through oxidation and reduction reactions. We recently demonstrated that by transferring electrons through such redox molecules (nature’s ‘‘electron’’ carriers), we can actuate gene expression in Escherichia coli (Tschirhart et al., Nat. Comm., published online January 17, 2017, https://dx.doi.org/ 10.1038/ncomms14030). Specifically, we used electrodes to oxidize pyocyanin, a redox active biological signal molecule that activates soxS-promoted gene expression in E. coli. In cells with an attenuated oxidative stress response regulon, we demonstrated a linear increase in gene expression with total electrical charge. By reversing the applied potential, we electronically turned ‘‘off’’ gene expression. Using this electrogenetic switch, we actuated several biological phenotypes, including motility and quorum sensing activity in nearby cells.
.we demonstrated a linear increase in gene expression with total electrical charge. What’s Next? This opens the door to a variety of functions, perhaps even connecting consumer electronics to biological systems that interact with humans. We envision devices that incorporate bacteria, perhaps even those engineered to make human pharmaceuticals.
Cell Systems 4, February 22, 2017 141
Cell Systems
Cell Systems Call Virtual Human Gut Microbiota Ronan Fleming and Ines Thiele, University of Luxembourg.
Principles The human gut microbiota is a metabolically active community of 10–100 trillion organisms comprising 500–1,000 species. The gut microbiota contributes to essential functions for the human host, such as food digestion, synthesis of essential amino acids and vitamins, protection against pathogens, and maturation of the host immune system. Changes in human gut microbial composition have been associated with many chronic diseases. So far, microbiome studies have provided valuable insights into which microbes live in our gut and where they are located in the gastrointestinal tract. A next challenge is to elucidate how these microbes modulate host metabolism, thereby moving beyond association to establish causation. To complement existing in vitro and in vivo tools, we generated in silico genome-scale metabolic reconstructions for 773 human gut bacteria (Magnu´sdo´ttir et. al., Nat. Biotechnol. 35, 81–89). The corresponding constraint-based computational microbial models serve as a molecular mechanistic framework for integration with, for example, personalized metagenomic data, to infer the metabolic capacity of the unique microbiota of each individual.
.we generated in silico genome-scale metabolic reconstructions for 773 human gut bacteria. What’s Next? Combining individualized microbiota models with a comprehensive molecular systems physiology model of human metabolism would enable the generation of mechanistic hypotheses on how microbial metabolic activity modulates host metabolism. Furthermore, the integration of personalized dietary information with such computational models would permit personalized exploration of the diet-gut-health axis and help to accelerate biomedicine by disentangling confounding factors.
142 Cell Systems 4, February 22, 2017
De Novo Design of Mammalian Promoters Joseph K. Cheng, University of Texas at Austin and Seattle Children’s Research Institute; and Hal S. Alper, The University of Texas at Austin
Principles Traditional mammalian promoter engineering emphasized cis-elements or DNA motifs (e.g., transcription factor binding sites, TFBSs) in regions 50 of the core promoter. Many of these regions were identified by dissecting key viral or endogenous genes, but do not give immediate clues of how to build synthetic promoters. Recently, we demonstrated a generalizable workflow toward the design of synthetic mammalian promoters built from endogenous transcription factors (Cheng and Alper, ACS Synth. Biol., 5, 1455–1465). Our premise was straightforward: genes that are well expressed in a desired condition have promoters that are enriched with TFBSs that enable expression in that state. Ultimately, we hypothesized that TFBSs enriched in strong endogenous promoters make good synthetic parts for creating strong synthetic promoters. We demonstrate that establishing synthetic promoters constructed with these TFBSs did, in fact, establish strong endogenous promoters that were comparable to commonly referenced viral- and endogenous-derived promoters (hCMV-IE and EEF1A1 gene, respectively).
.TFBSs enriched in strong endogenous promoters make good synthetic parts for creating strong synthetic promoters. What’s Next? This general workflow relies on existing technologies (expression profiling and DNA synthesis), thus enabling its usage in diverse cell types. The ability to precisely control gene expression is not only crucial for therapeutic applications such as immunotherapy, but also paves the way to engineer the metabolism of mammalian cell types, expanding the utility of these cells to be more than just protein factories.
Probing RAS Signaling with a Computationally Engineered Rheostat John Rose and Dustin Maly, University of Washington
Principles Engineered proteins that are temporally regulated by chemical genetic or optogenetic inputs are powerful tools for studying complex, dynamic cellular processes. These synthetic protein systems allow researchers to precisely manipulate specific nodes with high temporal resolution, absent the pleiotropic effects of natural stimuli. To date, the majority of these engineered systems rely on multi-component intermolecular regulation. Single-component, intramolecular regulatory systems are more attractive for many applications, but their development has been hampered by the difficulty that is inherent in engineering allostery. In Rose et al. (Nat. Chem, Biol. 13, 119– 126), we devised a framework for computationally guided design of intramolecular regulatory systems, which enabled the development of a RAS rheostat, chemically inducible activator of RAS (CIAR). CIAR rapidly and tunably activates RAS upon addition of a small molecule. Using CIAR, we examined the wiring of the RAS/ERKsignaling network. We found that RAS/ ERK-signaling kinetics differ between cell lines and that the RAF inhibitor vemurafenib amplifies the magnitude of this signal. Global phosphoproteomics further classified nodes based on their dynamic responses to direct RAS activation via CIAR or EGF stimulation. The generality of our computational approach was further demonstrated by the design of two intramolecularly regulated Rho-family guanine exchange factors.
.computationally guided design of intramolecular regulatory systems.enabled the development of a RAS rheostat. What’s Next? CIAR will enable quantitative interrogation of dynamic RAS-signaling networks in a wide array of model systems. Furthermore, we envision our computationally guided framework will expand researchers’ abilities to develop intramolecular regulatory systems for precisely controlling complex biological processes with novel engineered protein tools.
Cell Systems
Cell Systems Call Tunable Thermal Bioswitches for In Vivo Control of Bacterial Therapeutics Dan Piraner, Mohamad Abedi, and Mikhail Shapiro, California Institute of technology
Principles While our ability to engineer complex genetic circuits that orchestrate the behavior of therapeutic microbes has rapidly advanced, we still lack tools allowing us to communicate with microbial agents after their deployment inside host organisms, for example, to deliver instructions based on the agents’ location in the body. To provide such capabilities, we engineered tunable transcriptional bioswitches that enable noninvasive remote control over cell function deep inside living organisms (Piraner et al., Nat. Chem. Biol. 13, 75–80). We demonstrated the utility of these sensors in three in vivo bacterial therapy applications, in which bacteria were activated with spatiotemporal precision via focused ultrasound, modulated their gene expression in response to fever, and triggered cell death upon leaving the body.
.tunable transcriptional bioswitches that enable noninvasive control over cell function deep inside living organisms. What’s Next? In this work, we introduced a new expandable toolbox that allows for highly spatiotemporally restricted control of cell function, and we demonstrated the ability to engineer temperature-sensitive genetic circuits. These temperature sensors can be used in applications ranging from bacterial therapy to controlling protein expression in bioreactors. In addition, these sensors could be systematically transferred to other organisms where they can extend the capabilities of existing techniques for genetic control, such as optically or chemically triggered gene expression, into deep-tissue in vivo applications.
Sense Codon Reassignment Boosts Protein Chemistries Zhenling Cui, Sergey Mureev, and Kirill Alexandrov, University of Queensland
Principles Incorporation of unnatural amino acids (uAAs) via reassigned codons is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. So far, the site-selective incorporation of multiple uAAs into polypeptides was hampered by the limited number of available nonsense codons. We recently described a method for preparing an Escherichia coli in vitro translation system depleted of specific endogenous tRNA isoacceptors corresponding to split codon boxes. Such a system is dependent on the addition of synthetic tRNA(s) for the respective sense codon(s) for the restoration of its translational activity (Cui et al., ACS Synth Biol., published online December 14, 2016. https://dx.doi.org/10. 1021/acssynbio.6b00245). This allows multisite-selective incorporation of unnatural amino acids, and we successfully applied this approach to produce the calmodulin protein harboring FRET-forming fluorescent probes.
.in vitro translation system depleted of specific endogenous tRNAs.allows introduction of multiple unnatural amino acids into the predefined positions. What’s Next? The successful reassignment of AGG sense codon to uAAs using our newly developed technique demonstrates the feasibility of creating a potentially large number of reassignable orthogonal codons in an E. coli cell-free translation system. This potentially allows a significant expansion of the codons available for reassignment and allows introduction of multiple unnatural amino acids into the predefined positions. Furthermore, this approach can be transferred to eukaryotic cell-free systems, where the introduction of uAAs is lagging behind that of E. coli systems.
Cell Systems 4, February 22, 2017 143