Synthetic robust perfect adaptation achieved by negative feedback coupling with linear weak positive feedback.

Unlike their natural counterparts, synthetic genetic circuits are usually fragile in the face of environmental perturbations and genetic mutations. Several theoretical robust genetic circuits have been designed, but their performance under real-world conditions has not yet been carefully evaluated. Here, we designed and synthesized a new robust perfect adaptation circuit composed of two-node negative feedback coupling with linear positive feedback on the buffer node. As a key feature, the linear positive feedback was fine-tuned to evaluate its necessity. We found that the desired function was robustly achieved when genetic parameters were varied by systematically perturbing all interacting parts within the topology, and the necessity of the completeness of the topological structures was evaluated by destroying key circuit features. Furthermore, different environmental perturbances were imposed onto the circuit by changing growth rates, carbon metabolic strategies and even chassis cells, and the designed perfect adaptation function was still achieved under all conditions. The successful design of a robust perfect adaptation circuit indicated that the top-down design strategy is capable of predictably guiding bottom-up engineering for robust genetic circuits. This robust adaptation circuit could be integrated as a motif into more complex circuits to robustly implement more sophisticated and critical biological functions.

Paired dCas9 design as a nucleic acid detection platform for pathogenic strains.

The wide application of molecular beacon probes in specific DNA detection, especially in the fast prototyping of pathogen DNA detection kits in point-of-care diagnostics, has been hindered by the nonflexible choice of target sequences and the unstable fluorophore output. We developed an in vitro DNA detection system consisting of a pair of dCas9 proteins linked to split halves of luciferase, named the Paired dCas9 (PC) reporter. Co-localization of the reporter pair to a ~46 bp target sequence defined by two single guide RNAs (sgRNAs) activated luciferase which subsequently generated highly intensified luminescent signals. Combined with an array design and statistical analyses, the PC reporter system could be programmed to access sequence information across the entire genome of the pathogenic Mycobacterium tuberculosis H37Rv strain. These findings suggest great potential for the PC reporter in effective and affordable in vitro nucleic acid detection technologies. In this article we highlighted the systems design from our previous researchworkon the PC reporter (Zhang et al, 2015)with a focuson methodology.

A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts.

Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-ß-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-ß-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli.

Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host. A previous developed switch for butenoic acid fermentation is based on triclosan molecule as the FabI inhibitor in the fatty acid biosynthesis cycle. However, triclosan is toxic to human, which may limit its pharmaceutical application. Alternatively, we here purposed a nontoxic switch of carbon flux by harnessing recently developed CRISPR interference (CRISPRi) approach. In our work, we constructed a CRISPRi/dCpf1-mediated dynamic metabolic switch to separate the host growth and production phase via switching the expression of the fabI gene in fatty acid biosynthesis pathway. After optimizing the programmable targets, the CRISPRi-based switch boosted the titer of butenoic acid by 6-fold (1.41 g/L) in fed-batch fermentation. Our work supported that the CRISPRi/dCpf1 switch could replace triclosan-based switch as a nontoxic switch for butenoic acid production, and outcompeted the later switch in the biomass accumulation of the host cell. Moreover, the CRISPRi/dCpf1 system was integrated into the chromosome of the host to improve its genetic stability for long-term fermentation and other applications.Key Points• A programmable metabolic switch was developed to replace the toxic chemical switch to separate the growth phase and production phase of the butenoic acid.• The programmable CRISPRi/dCpf1 switch was efficiently and stably integrated into the host genome to increase their genetic stability during fermentation.• The optimized metabolic switch simultaneously increased the host biomass and butenoic acid titer, and solved the paradox of the competition between growth and production.

Genetic programs constructed from layered logic gates in single cells.

Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator-chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces.

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

Intermediate-sensor assisted push-pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli.

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push-pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an L-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with L-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).

Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

BACKGROUND: The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated. RESULTS: In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption. CONCLUSION: These results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces.

Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells.

RNAs and their encoded proteins intricately regulate diverse cell types and states within the human body. Dysregulated RNA expressions or mutations can lead to various diseased cell states, including tumorigenesis. Detecting and manipulating these endogenous RNAs offers significant promise for restoring healthy cell states and targeting tumors both in research and clinical contexts. This study presents an RNA-IN and RNA-OUT genetic circuit capable dynamically sensing and manipulating any RNA target in a programmable manner. The RNA-IN module employes a programmable CRISPR-associated protease (CASP) complex for RNA detection, while the RNA-OUT module utilizes an engineered protease-responsive dCas9-VPR activator. Additionally, the CASP module can detect point mutations by harnessing an uncovered dual-nucleotide synergistic switching effect within the CASP complex, resulting in the amplification of point-mutation signals from initially undetectable levels (1.5-fold) to a remarkable 94-fold. We successfully showcase the circuit's ability to rewire endogenous RNA-IN signals to activate endogenous progesterone biosynthesis pathway, dynamically monitor adipogenic differentiation of mesenchymal stem cells (MSCs) and the epithelial-to-mesenchmal trans-differentiation, as well as selective killing of tumor cells. The programmable RNA-IN and RNA-OUT circuit exhibits tremendous potential for applications in gene therapy, biosensing and design of synthetic regulatory networks.

A digital CRISPR-dCas9-based gene remodeling biocomputer programmed by dietary compounds in mammals.

CRISPR-dCas9 (dead Cas9 protein) technology, combined with chemical molecules and light-triggered genetic switches, offers customizable control over gene perturbation. However, these simple ON/OFF switches cannot precisely determine the sophisticated perturbation process. Here, we developed a resveratrol and protocatechuic acid-programmed CRISPR-mediated gene remodeling biocomputer (REPACRISPR) for conditional endogenous transcriptional regulation of genes in vitro and in vivo. Two REPACRISPR variants, REPACRISPRi and REPACRISPRa, were designed for the logic control of gene inhibition and activation, respectively. We successfully demonstrated the digital computations of single or multiplexed endogenous gene transcription by using REPACRISPRa. We also established mathematical models to predict the dose-responsive transcriptional levels of a target endogenous gene controlled by REPACRISPRa. Moreover, high levels of endogenous gene activation in mice mediated by the AND logic gate demonstrated computational control of CRISPR-dCas9-based epigenome remodeling in mice. This CRISPR-based biocomputer expands the synthetic biology toolbox and can potentially advance gene-based precision medicine. A record of this paper's transparent peer review process is included in the supplemental information.

Quantitative Characterization of Gene Regulatory Circuits Associated With Fungal Secondary Metabolism to Discover Novel Natural Products.

Microbial genetic circuits are vital for regulating gene expression and synthesizing bioactive compounds. However, assessing their strength and timing, especially in multicellular fungi, remains challenging. Here, an advanced microfluidic platform is combined with a mathematical model enabling precise characterization of fungal gene regulatory circuits (GRCs) at the single-cell level. Utilizing this platform, the expression intensity and timing of 30 transcription factor-promoter combinations derived from two representative fungal GRCs, using the model fungus Aspergillus nidulans are determined. As a proof of concept, the selected GRC combination is utilized to successfully refactor the biosynthetic pathways of bioactive molecules, precisely control their production, and activate the expression of the silenced biosynthetic gene clusters (BGCs). This study provides insights into microbial gene regulation and highlights the potential of platform in fungal synthetic biology applications and the discovery of novel natural products.

Improving cooperativity of transcription activators by oligomerization domains in mammalian cells.

Cooperative activation is critical for the applications of synthetic biology in mammalian cells. In this study, we have developed cooperative transcription factor by fusing oligomerization domain in mammalian cells. Firstly, we demonstrated that two oligomerized domains (CI434 and CI) successfully improved transcription factor cooperativity in bacterial cells but failed to increase cooperativity in mammalian cells, possibly because the additional mammalian activation domain disrupted their oligomerization capability. Therefore, we chose a different type of oligomerized domain (CarHC), whose ability to oligomerize is not dependent on its C-terminal domains, to fuse with a transcription factor (RpaR) and activation domain (VTR3), forming a potential cooperative transcription activator RpaR-CarH-VTR3 for mammalian regulatory systems. Compared with RpaR-VTR3, the cooperativity of RpaR-CarH-VTR3 was significantly improved with higher Hill coefficient and a narrower input range in the inducible switch system in mammalian cells. Moreover, a mathematical model based on statistical mechanics model was developed and the simulation results supported the hypothesis that the tetramer of the CarH domain in mammalian cells was the reason for the cooperative capacity of RpaR-CarH-VTR3.

The Topological Characteristics of Biological Ratio-Sensing Networks.

Ratio sensing is a fundamental biological function observed in signal transduction and decision making. In the synthetic biology context, ratio sensing presents one of the elementary functions for cellular multi-signal computation. To investigate the mechanism of the ratio-sensing behavior, we explored the topological characteristics of biological ratio-sensing networks. With exhaustive enumeration of three-node enzymatic and transcriptional regulatory networks, we found that robust ratio sensing was highly dependent on network structure rather than network complexity. Specifically, a set of seven minimal core topological structures and four motifs were deduced to be capable of robust ratio sensing. Further investigations on the evolutionary space of robust ratio-sensing networks revealed highly clustered domains surrounding the core motifs which suggested their evolutionary plausibility. Our study revealed the network topological design principles of ratio-sensing behavior and provided a design scheme for constructing regulatory circuits with ratio-sensing behavior in synthetic biology.

Corrigendum to "Improving cooperativity of transcription activators by oligomerization domains in mammalian cells" [Synth Syst Biotechnol 8 (1) (2023) 114-120].

[This corrects the article DOI: 10.1016/j.synbio.2022.12.003.].

Quantitative characterization of filamentous fungal promoters on a single-cell resolution to discover cryptic natural products.

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods. Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution. Using this quantitative method, we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format. The strengths of identified promoters covered a 37-fold range by flow cytometry. PzipA and PsltA were identified as the strongest promoters, which were 2.9- and 1.5-fold higher than that of the commonly used constitutive promoter PgpdA. Thus, we applied PzipA and PsltA to activate the silent nonribosomal peptide synthetase gene Afpes1 from Aspergillus fumigatus in its native host and the heterologous host A. nidulans. The metabolic products of Afpes1 were identified as new cyclic tetrapeptide derivatives, namely, fumiganins A and B. Our method provides an innovative strategy for natural product discovery in fungi.

Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system.

Context-dependency of mammalian transcriptional elements has hindered the quantitative investigation of multigene expression stoichiometry and its biological functions. Here, we describe a host- and local DNA context-independent transcription system to gradually fine-tune single and multiple gene expression with predictable stoichiometries. The mammalian transcription system is composed of a library of modular and programmable promoters from bacteriophage and its cognate RNA polymerase (RNAP) fused to a capping enzyme. The relative expression of single genes is quantitatively determined by the relative binding affinity of the RNAP to the promoters, while multigene expression stoichiometry is predicted by a simple biochemical model with resource competition. We use these programmable and modular promoters to predictably tune the expression of three components of an influenza A virus-like particle (VLP). Optimized stoichiometry leads to a 2-fold yield of intact VLP complexes. The host-independent orthogonal transcription system provides a platform for dose-dependent control of multiple protein expression which may be applied for advanced vaccine engineering, cell-fate programming and other therapeutic applications.

Synthetic Genetic Elements, Devices, and Systems.

Since the beginning of life on Earth, over the course of 3 to 4 billion years, nature has created vast quantities of genetic elements [...].

Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous Streptomyces Hosts.

Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 µg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers.