Rate of environmental change determines stress response specificity

Cells use general stress response pathways to activate diverse target genes in response to a variety of stresses. However, general stress responses coexist with more specific pathways that are activated by individual stresses, provoking the fundamental question of whether and how cells control the generality or specificity of their response to a particular stress. Here we address this issue using quantitative time-lapse microscopy of the Bacillus subtilis environmental stress response, mediated by σ^B. We analyzed σ^B activation in response to stresses such as salt and ethanol imposed at varying rates of increase. Dynamically, σ^B responded to these stresses with a single adaptive activity pulse, whose amplitude depended on the rate at which the stress increased. This rate-responsive behavior can be understood from mathematical modeling of a key negative feedback loop in the underlying regulatory circuit. Using RNAseq we analyzed the effects of both rapid and gradual increases of ethanol and salt stress across the genome. Because of the rate responsiveness of σ^B activation, salt and ethanol regulons overlap under rapid, but not gradual, increases in stress. Thus, the cell responds specifically to individual stresses that appear gradually, while using σ^B to broaden the cellular response under more rapidly deteriorating conditions. Such dynamic control of specificity could be a critical function of other general stress response pathways.     

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids.

Most metabolic engineering strategies for the production of valuable chemicals utilize the cytosol as a one-pot reaction vessel, where multiple enzymes are introduced to catalyze long reaction cascades (1). However, this approach frequently results in poor yields, formation of undesirable by-products, or toxicity (2, 3), because cellular metabolism is an intricate mesh of reactions involving extensive cross-talk and elaborate regulatory mechanisms. Living organisms have devised solutions to overcome similar challenges by confining pathways within intracellular compartments to streamline reaction cascades and shield intermediates from competing pathways (4). Thus, nature’s compartmentalization strategy can provide inspiration for solving metabolic engineering challenges (5).Monoterpenoids, monoterpene indole alkaloids, and cannabinoids are groups of specialized metabolites valued for their fragrant and therapeutic properties. Due to a sizeable and rapidly expanding world market [collectively currently exceeding 10 billion USD (6, 7)], sourcing these molecules from nature or deriving them from petrochemicals is no longer sustainable, prompting efforts for their production in engineered microorganisms (8–12). The biosynthesis of all these compound groups shares a common building block, the universal 10-carbon isoprenoid precursor geranyl diphosphate (GPP) (13). However, synthesis of GPP-derived compounds in the industrial workhorse Saccharomyces cerevisiae has been particularly challenging due to the difficulty in creating a sufficiently large pool of GPP (10). This is the result of a strong competition for GPP by the native sterol biosynthesis pathway (10), which is essential for growth (Fig. 1). In yeast, GPP is only synthesized as an intermediate en route to farnesyl diphosphate (FPP), the precursor of sterols, and no other yeast metabolite is produced from GPP. Yeast cells employ a highly efficient cytoplasmic enzyme, Erg20p, which functions as a bifunctional synthase that condenses isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) first to GPP and subsequently to FPP (Fig. 1). As a result, once synthesized, GPP is rapidly converted to FPP by Erg20p, not allowing high enough fluxes to heterologous pathways converting GPP to desirable high-value metabolites (10, 14). Although several innovative strategies have been employed to overcome this limitation, such as engineering Erg20p stability (15) and specificity (14), or establishment of an orthogonal pathway based on an isomeric substrate (10), synthesis of 10-carbon isoprenoids in yeast is still markedly less efficient than other isoprenoids, whose production has reached industrial levels (16–18). For example, the sesquiterpenes farnesene and amorphadiene are being produced in titers that exceed 134 g/L and 40 g/L, respectively, while the highest reported monoterpene titers in yeast currently do not exceed 0.9 g/L for limonene (19) and 1.68 g/L for geraniol (20).Open in a separate windowFig. 1.Engineering a GPP-synthesizing microfactory in the yeast peroxisome. Conventional production of GPP-derived compounds in yeast relies on the endogenous cytosolic MVA pathway (shown in black). Here, we build an insulated pathway for GPP synthesis in the peroxisome by targeting the complete MVA pathway in the peroxisome (shown in green) and harvesting peroxisomal acetyl-CoA. Introduction of specific monoterpene synthases (SeCamS, SpSabS, ObGerS, MsLimS, ClLimS, PtPinS, ObGerS) in the peroxisome together with the GPP synthase Erg20p(N127W) (also shown in green) leads to strong increase in the production of the corresponding monoterpene scaffolds compared to the cytosol. When the monoterpene synthase and Erg20p(N127W) are targeted in the peroxisome in the absence of a peroxisomal MVA pathway, IPP and DMAPP coming from the cytosol (indicated by black arrows crossing the peroxisomal membrane) can still support synthesis of various monoterpenes, albeit at a lower level. Further oxidation of monoterpene scaffolds by cytochrome P450 enzymes (CrG8OH and MsLimH, also shown in green) residing at the ER provide precursors of high-value molecules, such as monoterpene indole alkaloids and menthol. The peroxisomal GPP pool can also be used for OA prenylation leading to CBGA, the common precursor of various cannabinoids. For further explanation of the steps and pathways, please refer to the legend embedded in the figure. HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA A.The challenge faced in the production of GPP-derived compounds can be addressed by the same strategy used by eukaryotic cells to solve similar issues within their own metabolism. Organelles, such as mitochondria, plastids, and peroxisomes, are exploited to isolate intermediates from competing pathways, shield enzymes from inhibitors, and protect the rest of the cell from toxic compounds. Thus, an attractive alternative would be to establish GPP synthesis in a yeast organelle and cocompartmentalize the heterologous GPP-utilizing enzymes. Shielding GPP from a strongly competing pathway would allow a large enough GPP pool to build up and give the opportunity to enzymes with a lesser catalytic efficiency than Erg20p (k_cat/K_M for GPP of 1.4 × 10⁵ M⁻¹⋅s⁻¹) (14), such as monoterpene synthases (k_cat/K_M in the range of 10³ to 10⁴ M⁻¹⋅s⁻¹) (10), to access the substrate.To implement this strategy, we focused on the peroxisomes. In S. cerevisiae, the peroxisomes are a suitable target for organellar engineering because, in most culture conditions, they are not essential for cell viability (21) and their number and size can be modified in various ways to better fit the needs of the engineered pathway (22, 23). The yeast peroxisomes are the sites where β-oxidation of fatty acids takes place, creating a pool of acetyl-CoA that can be harvested by heterologous pathways, such as the mevalonate (MVA) pathway that provides the isoprenoid precursors IPP and DMAPP (23, 24). Moreover, the peroxisomes have a single-layer membrane that allows a large number of low molecular weight compounds to travel across, either passively or through channel proteins, such as Pxmp2 (25). This can be advantageous for engineering the spatial confinement of substrates and enzymes, without restricting the exchange of precursors and products with the remaining of the cell or requiring the introduction of dedicated transporters (26). The peroxisomes are also detoxifying organelles that can handle and sequester more toxic molecules from the rest of the cell (27). Although other organelles, such as the mitochondria, could be used for a similar compartmentalization strategy, these are formed by double membranes, which can create a strong barrier to substrates or products, requiring considerable transport engineering for optimal performance. Furthermore, mitochondria carry out essential functions for cell viability and, as such, cannot be extensively engineered without affecting yeast fitness (28). The above-mentioned favorable characteristics of the peroxisomes have led to their successful application in the production of medium-chain fatty alcohols, alkanes, olefins (23, 24), or alkaloids (29) in S. cerevisiae, penicillin in Hansenula polymorpha (30), polyhydroxyalkanoates, and polylactic acid in Pichia pastoris and Yarrowia lipolytica (26, 31).In this study, we demonstrate that the yeast peroxisomes can be advantageously repurposed for the production of GPP-derived molecules. We introduce a complete MVA pathway into the peroxisomes to establish efficient GPP-synthesizing microfactories that harvest the peroxisomal acetyl-CoA pool to produce a wide variety of GPP-derived compounds. We show that peroxisomal production can be used as a general strategy for the synthesis of monoterpene scaffolds by demonstrating that it results in important improvements in the production of several different compounds, including (R)-(+)-limonene, (S)-(-)-limonene, α-pinene, sabinene, camphene, and geraniol. Additionally, we show that the improved supply of monoterpene precursors, and likely the proximity of the peroxisomes to the endoplasmic reticulum (ER) (32–34), improve cytochrome P450-mediated oxidation of monoterpenes. This enables establishing the next step toward menthol and monoterpene indole alkaloid biosynthesis, through efficient production of trans-isopiperitenol and 8-hydroxygeraniol, respectively. The applicability of the peroxisome in the production of high-value molecules is exemplified even further by the synthesis of cannabigerolic acid (CBGA), the common precursor of cannabinoids. Finally, we evaluate this strategy in fed-batch fermentation and achieve titers of commercially important monoterpenes that bring them closer to industrial production without noticeable effects in strain fitness or viability. Our findings highlight peroxisomal engineering as an efficient strategy for the biotechnological production of high-value isoprenoids.     

Synthetic spatially graded Rac activation drives cell polarization and movement

Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.     

Functional organization of the yeast proteome by a yeast interactome map

It is hoped that comprehensive mapping of protein physical interactions will facilitate insights regarding both fundamental cell biology processes and the pathology of diseases. To fulfill this hope, good solutions to 2 issues will be essential: (i) how to obtain reliable interaction data in a high-throughput setting and (ii) how to structure interaction data in a meaningful form, amenable to and valuable for further biological research. In this article, we structure an interactome in terms of predicted permanent protein complexes and predicted transient, nongeneric interactions between these complexes. The interactome is generated by means of an associated computational algorithm, from raw high-throughput affinity purification/mass spectrometric interaction data. We apply our technique to the construction of an interactome for Saccharomyces cerevisiae, showing that it yields reliability typical of low-throughput experiments from high-throughput data. We discuss biological insights raised by this interactome including, via homology, a few related to human disease.     

The use of oscillatory signals in the study of genetic networks

The structure of a genetic network is uncovered by studying its response to external stimuli (input signals). We present a theory of propagation of an input signal through a linear stochastic genetic network. We found that there are important advantages in using oscillatory signals over step or impulse signals and that the system may enter into a pure fluctuation resonance for a specific input frequency.     

Conversion of CO2 into organic acids by engineered autotrophic yeast

The increase of CO₂ emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO₂ as a feedstock gains an extensive importance to establish CO₂-neutral or CO₂-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO₂ as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. ¹³C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO₂ as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L⁻¹, while balancing the expression of key genes and modifying screening conditions led to 2 g L⁻¹ itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO₂ uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO₂, to support sustainability of industrial bioprocesses.

Between 2011 and 2020 the annual average CO₂ emission due to human activity exceeded 38 gigatons, of which around 22 gigatons are removed again from the atmosphere to terrestrial and ocean CO₂ sinks. This imbalance, mostly due to combustion of fossil fuels, causes the steady increase in CO₂ levels in the atmosphere which is one of the primary reasons for the climate crisis our planet is facing today (1).Biologically produced fuels and commodity chemicals bear the potential to counteract this deleterious development, but the most common feedstocks used for bioproduction, such as glucose, sucrose and starch rely on agricultural production and are bearing the risk to threaten food security. Using autotrophic microorganisms as production platforms exploits the potential of CO₂ itself as an alternative carbon source. In nature, autotrophic microorganisms play a major role in CO₂ fixation by fixing 200 gigatons of CO₂ every year (2). However, the rates of most natural microbial CO₂ fixing pathways are low: the photosynthetic efficiency in cyanobacteria is limited to 1 to 2% which makes industrial processes using photoautotrophs economically less feasible (3). Chemoautotrophs may overcome this barrier as their energy harvesting processes are more efficient than light harvesting of photoautotrophs.One well-known autotroph that is being developed for biological production of materials is Cupriavidus necator (formerly known as Ralstonia eutropha). By harvesting energy with a controlled Knallgas reaction these bacteria assimilate CO₂ via the Calvin-Benson-Bessham (CBB) cycle. Besides naturally produced polyhydroxyalkanoates (PHA), metabolic pathway engineering enabled the production of several other chemicals (4–7).Several chemolithotrophic bacteria were demonstrated as production hosts for various chemicals such as ethanol, 2,3-butanediol, butanol, isopropanol, acetone, or isobutyric acid via natural carbon assimilation pathways (8–11). In addition to natural CO₂ fixing microorganisms, implementation of heterologous CO₂ fixing pathways to heterotrophic microorganisms like Myceliophthora thermophila provided a mixotrophic strain that is able to produce malic acid with a higher yield compared to the parent strain in which only the reductive tricarboxylic acid (TCA) cycle is used for the production (12).Natural chemoautotrophs have a large potential to convert CO₂ to chemicals. However, they are often recalcitrant to genetic editing, have complex nutrient demands, or may require complex process technological solutions like the transfer of gaseous substrates and energy sources. To circumvent some of these limitations well established prokaryotic and eukaryotic production hosts have been engineered to assimilate CO₂. The bacterial workhorse Escherichia coli and the yeast Komagataella phaffii (Pichia pastoris) were provided with the CBB cycle, enabling them to assimilate CO₂ by using formate or methanol, respectively, as energy sources. Both engineered microorganisms can grow sustainably with CO₂ as carbon source (13, 14). Conceptually formate and methanol are regarded as sustainable feedstocks for biotechnology when they are derived from CO₂ by hydrogenation or electrochemical reduction (15).To make an impact on the global CO₂ household such autotrophic processes need to convert CO₂ into bulk products. Besides ethanol, short chain organic acids are the second largest group of chemicals manufactured by industrial biotechnology. The market of biologically produced organic acids is expected to reach more than $36 billion by 2026 (16). The annual bioproduction of some of the key organic acids (citric, acetic, lactic, succinic acid) is more than 12 million metric tons (17–20). Recently, itaconic acid has also gained attention as a promising chemical building block with an estimated market increase to 170 kilotons per year and $260 million in 2025 (21). Lactic and itaconic acid are feedstocks for polymer production so that they, and other biobased commodity chemicals compete for the annual polymer production of more than 300 million tons, a volume that denotes a significant impact on the global CO₂ balance.Here, we set out to evaluate if the autotrophic K. phaffii strain can be used as a platform for organic acid production. Synthetic autotrophy was introduced to K. phaffii by converting the native peroxisomal methanol assimilation pathway, the xylulose monophosphate (XuMP) cycle, into the CBB cycle (13). To achieve that, the formaldehyde assimilating enzyme dihydroxyacetone synthase (DAS) was replaced by a bacterial ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and phosphoribulose kinase (PRK) from spinach was added to supply ribulose bisphosphate from XuMP precursors. Four yeast glycolytic enzymes were targeted to peroxisomes to close the CBB cycle to glyceraldehyde 3-phosphate (G3P), both as an intermediate and the product of the assimilation cycle (Fig. 1 A and B). In the present work, using modular synthetic biology tools, we implemented the genes for lactic and itaconic acid synthesis plus accessory genes into the K. phaffii genome and demonstrate that the autotrophic K. phaffii strain is capable of producing organic acids solely from CO₂ as carbon source.Open in a separate windowFig. 1.Expression of cadA and ldhL enables organic acid production in synthetic autotrophic K. phaffii. (A–D) Schematic pathways. (A) In wild-type K. phaffii methanol is oxidized to formaldehyde (black arrow) and assimilated in the XuMP cycle (orange arrows) or dissimilated to CO₂, respectively (purple arrow). (B) synthetic autotrophy in K. phaffii: the native assimilatory branch of methanol utilization was interrupted by deleting DAS1 and DAS2 (dashed gray line). AOX1 was knocked out to reduce the rate of formaldehyde formation which could be toxic to the cells. RuBisCO and PRK were integrated to complete a functional CBB cycle (green arrows). Additionally, two bacterial chaperones, groEL and groES, were overexpressed to assist the folding of RuBisCO. TDH3, PGK1, TKL1, TPI1 carrying each a peroxisomal targeting signal were overexpressed to assure the localization of the entire CBB cycle in peroxisomes. More details about the engineering strategy can be found in ref. (13). (C) Itaconic acid (red) and (D) lactic acid production (blue), (E) growth profiles, and (F) organic acid production profiles of the producing strains and the control. Time axis corresponds to the production phase under autotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±). 3PG: 3-phosphoglycerate, AcCoA: acetyl-coenzyme A, AOX1 and AOX2: alcohol oxidase 1 and 2, cadA: cis-aconitate decarboxylase, CBB cycle: Calvin-Benson-Bassham cycle, CISA_c: cytosolic cis-aconitate, CISA_m: mitochondrial cis-aconitate, DAS1 and DAS2: dihydroxyacetone synthase 1 and 2, DHA: dihydroxyacetone, FAL: formaldehyde, G3P: glyceraldehyde 3-phosphate, ITA: itaconic acid, LA: lactic acid, ldhL: L-lactate dehydrogenase, MeOH: methanol, mttA: mitochondrial tricarboxylic acid transporter, NAD⁺/NADH: nicotinamide adenine dinucleotide, PRK: phosphoribulokinase, PYR: pyruvate, RuBP: ribulose 1,5-bisphosphate, RuBisCO: ribulose 1,5-bisphosphate carboxylase/oxygenase, Xu5P: xylulose 5-phosphate, XuMP cycle: xylulose monophosphate cycle.     

Pairwise network mechanisms in the host signaling response to coxsackievirus B3 infection

Signal transduction networks can be perturbed biochemically, genetically, and pharmacologically to unravel their functions. But at the systems level, it is not clear how such perturbations are best implemented to extract molecular mechanisms that underlie network function. Here, we combined pairwise perturbations with multiparameter phosphorylation measurements to reveal causal mechanisms within the signaling network response of cardiomyocytes to coxsackievirus B3 (CVB3) infection. Using all possible pairs of six kinase inhibitors, we assembled a dynamic nine-protein phosphorylation signature of perturbed CVB3 infectivity. Cluster analysis of the resulting dataset showed repeatedly that paired inhibitor data were required for accurate data-driven predictions of kinase substrate links in the host network. With pairwise data, we also derived a high-confidence network based on partial correlations, which identified phospho-IκBα as a central “hub” in the measured phosphorylation signature. The reconstructed network helped to connect phospho-IκBα with an autocrine feedback circuit in host cells involving the proinflammatory cytokines, TNF and IL-1. Autocrine blockade substantially inhibited CVB3 progeny release and improved host cell viability, implicating TNF and IL-1 as cell autonomous components of CVB3-induced myocardial damage. We conclude that pairwise perturbations, when combined with network-level intracellular measurements, enrich for mechanisms that would be overlooked by single perturbants.     

The main auxin biosynthesis pathway in Arabidopsis

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.     

Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation

Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.

Artificial cells are engineered mimics of biological cells, constructed from the bottom up by bringing together defined molecular building blocks (1). They are designed to replicate the form, function, and behaviors of natural cells, and most often are based on enclosed compartments that contain biomolecular species responsible for imparting cell-like features (compartmentalization, sense/response, communication, etc.) (2, 3). In addition to being used as simplified cell models to decipher the rules of life through an “understanding by building” approach (4), a major motivation behind artificial cell research is their potential to act as devices that can be used in biomedical and biotechnological applications (5–7).There are several advantages associated with artificial cells compared with genetically engineered biological cells that are the preserve of top-down synthetic biology. This includes the ability to incorporate nonbiological functional components, reduced regulatory and biosafety considerations, and the removal of cell-burden limitations that arise when engineered cellular functions exist alongside native ones (8). However, perhaps the major attraction of artificial cells is that they only contain the minimal components required to perform their function and can thus be composed of only a small number of molecular species. It is therefore easier to engineer them to have user-defined features, so they are highly controllable and programmable. However, their reduced complexity means that they cannot currently match the metabolic, regulatory, and behavioral sophistication of their biological counterparts. Processes in biological cells may thus be more robust, with cells able to perform their functions even in the presence of perturbations or changes in their milieu (9), which is often the basis of their use in many applications (10).The programmability of artificial cells and the technological potential of biological cells have led to increased efforts at coupling the two together, and in the process, accumulating the advantages associated with both (11). This can be achieved by either forming hybrid living/artificial cells (12–14) or engineering communication routes between populations of living cells (bacteria or eukaryotes) and artificial ones (15–20). In so doing, not only could the extensive genetic modification of cells be bypassed (as the artificial cells are the engineered species), but also more complex functions could be reconstituted when both types of cells are incorporated into the same system.Integrating artificial and biological cells requires establishing communication pathways between them, for example, through quorum sensing using autoinducer molecules (21, 22). This approach was exploited to expand the sensory range of bacteria through the construction of artificial cells that operated as chemical translators that could sense a small molecule and release an inducer molecule that bacteria could respond to (23). Similarly, robust artificial cells were designed to act as sensor or reporter modules depending on the DNA program present in the artificial or bacterial cell (24). In recent years, engineered communication has been extended to artificial-eukaryotic cell networks (17, 25–27).Nevertheless, the current systems rely on chemical triggers, while the use of physical stimuli remains mostly unexplored. We intended to remedy this by engineering stimuli-responsive artificial cells which can communicate with bacteria.In so doing, stimuli-responsive artificial cells would constitute distributed “artificial organelles,” which are not encapsulated within living cells but endow bacteria with novel responsiveness to physical stimuli. This would also provide artificial/living hybrid cell systems with a control step to allow external activation by an end user on demand. Designing a library of stimuli-responsive platforms entails the next breakthrough in cellular bionics, since controlling cell behaviors using artificial cells as intermediaries would enable processes to take place in a temporally controlled manner, in a defined location, and in response to specific stimuli. There have been several examples of stimuli-responsive population communication operating between synthetic cell populations, including communication between cell-like compartments in synthetic tissues using a light-regulated DNA promoter (28), through divalent cation chelators (29), or using a photolabile DNA linker (30). In a recent exciting preprint, a light-activated DNA template was used to control communication between synthetic and bacterial cells using ultraviolet (UV) light (31). Nevertheless, to realize the incorporation of hybrid artificial/living cell systems in applications such as therapeutics, bioremediation, and biosensing, remote-controlled cell mimics able to communicate with a wide range of biological cells, using different stimuli, need to be further developed (32). Herein, we address this gap by designing a generalizable vesicle-based artificial cell platform capable of compartmentalizing content, sensing a physical cue, and releasing chemical messengers that can then activate a DNA program in Escherichia coli.To do this, we developed and exploited two types of synthetic cell compartments, one of which was light responsive and the other thermoresponsive. These house inducer molecules with them, which are ordinarily shielded from surrounding bacteria. Only upon encountering the relevant stimuli do these molecules get released and activate protein expression in the living cells. This allowed us to control protein expression in cells using artificial cells and intermediaries that translate the stimuli (light and heat) into a chemical signal that the cells respond to.     

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.     

Inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5′-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for “green” manufacturing processes.     

Derivation of Ca2+ signals from puff properties reveals that pathway function is robust against cell variability but sensitive for control

Ca(2+) is a universal second messenger in eukaryotic cells transmitting information through sequences of concentration spikes. A prominent mechanism to generate these spikes involves Ca(2+) release from the endoplasmic reticulum Ca(2+) store via inositol 1,4,5-trisphosphate (IP(3))-sensitive channels. Puffs are elemental events of IP(3)-induced Ca(2+) release through single clusters of channels. Intracellular Ca(2+) dynamics are a stochastic system, but a complete stochastic theory has not been developed yet. We formulate the theory in terms of interpuff interval and puff duration distributions because, unlike the properties of individual channels, they can be measured in vivo. Our theory reproduces the typical spectrum of Ca(2+) signals like puffs, spiking, and bursting in analytically treatable test cases as well as in more realistic simulations. We find conditions for spiking and calculate interspike interval (ISI) distributions. Signal form, average ISI and ISI distributions depend sensitively on the details of cluster properties and their spatial arrangement. In contrast to that, the relation between the average and the standard deviation of ISIs does not depend on cluster properties and cluster arrangement and is robust with respect to cell variability. It is controlled by the global feedback processes in the Ca(2+) signaling pathway (e.g., via IP(3)-3-kinase or endoplasmic reticulum depletion). That relation is essential for pathway function because it ensures frequency encoding despite the randomness of ISIs and determines the maximal spike train information content. Hence, we find a division of tasks between global feedbacks and local cluster properties that guarantees robustness of function while maintaining sensitivity of control of the average ISI.     

Tailored fatty acid synthesis via dynamic control of fatty acid elongation

Medium-chain fatty acids (MCFAs, 4–12 carbons) are valuable as precursors to industrial chemicals and biofuels, but are not canonical products of microbial fatty acid synthesis. We engineered microbial production of the full range of even- and odd-chain–length MCFAs and found that MCFA production is limited by rapid, irreversible elongation of their acyl-ACP precursors. To address this limitation, we programmed an essential ketoacyl synthase to degrade in response to a chemical inducer, thereby slowing acyl-ACP elongation and redirecting flux from phospholipid synthesis to MCFA production. Our results show that induced protein degradation can be used to dynamically alter metabolic flux, and thereby increase the yield of a desired compound. The strategy reported herein should be widely useful in a range of metabolic engineering applications in which essential enzymes divert flux away from a desired product, as well as in the production of polyketides, bioplastics, and other recursively synthesized hydrocarbons for which chain-length control is desired.     

From the Cover: Kinematic self-replication in reconfigurable organisms

All living systems perpetuate themselves via growth in or on the body, followed by splitting, budding, or birth. We find that synthetic multicellular assemblies can also replicate kinematically by moving and compressing dissociated cells in their environment into functional self-copies. This form of perpetuation, previously unseen in any organism, arises spontaneously over days rather than evolving over millennia. We also show how artificial intelligence methods can design assemblies that postpone loss of replicative ability and perform useful work as a side effect of replication. This suggests other unique and useful phenotypes can be rapidly reached from wild-type organisms without selection or genetic engineering, thereby broadening our understanding of the conditions under which replication arises, phenotypic plasticity, and how useful replicative machines may be realized.

Like the other necessary abilities life must possess to survive, replication has evolved into many diverse forms: fission, budding, fragmentation, spore formation, vegetative propagation, parthenogenesis, sexual reproduction, hermaphroditism, and viral propagation. These diverse processes however share a common property: all involve growth within or on the body of the organism. In contrast, a non–growth-based form of self-replication dominates at the subcellular level: molecular machines assemble material in their external environment into functional self-copies directly, or in concert with other machines. Such kinematic replication has never been observed at higher levels of biological organization, nor was it known whether multicellular systems were even capable of it.Despite this lack, organisms do possess deep reservoirs of adaptive potential at all levels of organization, allowing for manual or automated interventions that deflect development toward biological forms and functions different from wild type (1), including the growth and maintenance of organs independent of their host organism (2–4), or unlocking regenerative capacity (5–7). Design, if framed as morphological reconfiguration, can reposition biological tissues or redirect self-organizing processes to new stable forms without recourse to genomic editing or transgenes (8). Recent work has shown that individual, genetically unmodified prospective skin (9) and heart muscle (10) cells, when removed from their native embryonic microenvironments and reassembled, can organize into stable forms and behaviors not exhibited by the organism from which the cells were taken, at any point in its natural life cycle. We show here that if cells are similarly liberated, compressed, and placed among more dissociated cells that serve as feedstock, they can exhibit kinematic self-replication, a behavior not only absent from the donating organism but from every other known plant or animal. Furthermore, replication does not evolve in response to selection pressures, but arises spontaneously over 5 d given appropriate initial and environmental conditions.     

Gravity-induced PIN transcytosis for polarization of auxin fluxes in gravity-sensing root cells

Auxin is an essential plant-specific regulator of patterning processes that also controls directional growth of roots and shoots. In response to gravity stimulation, the PIN3 auxin transporter polarizes to the bottom side of gravity-sensing root cells, presumably redirecting the auxin flux toward the lower side of the root and triggering gravitropic bending. By combining live-cell imaging techniques with pharmacological and genetic approaches, we demonstrate that PIN3 polarization does not require secretion of de novo synthesized proteins or protein degradation, but instead involves rapid, transient stimulation of PIN endocytosis, presumably via a clathrin-dependent pathway. Moreover, gravity-induced PIN3 polarization requires the activity of the guanine nucleotide exchange factors for ARF GTPases (ARF-GEF) GNOM-dependent polar-targeting pathways and might involve endosome-based PIN3 translocation from one cell side to another. Our data suggest that gravity perception acts at several instances of PIN3 trafficking, ultimately leading to the polarization of PIN3, which presumably aligns auxin fluxes with gravity vector and mediates downstream root gravitropic response.     

Rapid and robust signaling in the CsrA cascade via RNA–protein interactions and feedback regulation

Bacterial survival requires the rapid propagation of signals through gene networks during stress, but how this is achieved is not well understood. This study systematically characterizes the signaling dynamics of a cascade of RNA–protein interactions in the CsrA system, which regulates stress responses and biofilm formation in Escherichia coli. Noncoding RNAs are at the center of the CsrA system; target mRNAs are bound by CsrA proteins that inhibit their translation, CsrA proteins are sequestered by CsrB noncoding RNAs, and the degradation of CsrB RNAs is increased by CsrD proteins. Here, we show using in vivo experiments and quantitative modeling that the CsrA system integrates three strategies to achieve rapid and robust signaling. These strategies include: (i) the sequestration of stable proteins by noncoding RNAs, which rapidly inactivates protein activity; (ii) the degradation of stable noncoding RNAs, which enables their rapid removal; and (iii) a negative-feedback loop created by CsrA repression of CsrD production, which reduces the time for the system to achieve steady state. We also demonstrate that sequestration in the CsrA system results in signaling that is robust to growth rates because it does not rely on the slow dilution of molecules via cell division; therefore, signaling can occur even during growth arrest induced by starvation or antibiotic treatment.