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.

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.

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.

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.

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.

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.].

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 [...].

Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces.

The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic Streptomyces strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter Pecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal Pecf17 promoter was determined, and a Pecf17 promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S. venezuelae ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 ± 15.3  µ g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S. venezuelae ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces albus J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 µg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse Streptomyces hosts.

Mitigating Host Burden of Genetic Circuits by Engineering Autonegatively Regulated Parts and Improving Functional Prediction.

Mitigating unintended interferences between circuits and host cells is key to realize applications of synthetic regulatory systems both for bacteria and mammalian cells. Here, we demonstrated that growth burden and circuit dysregulation occurred in a concentration-dependent manner for specific transcription factors (CymR*/CymR) in E.coli, and direct negative feedback modules were able to control the concentration of CymR*/CymR, mitigate growth burden, and restore circuit functions. A quantitative design scheme was developed for circuits embedded with autorepression modules. Four key parameters were theoretically identified to determine the performance of autoregulated switches and were experimentally modified by fine-tuning promoter architectures and cooperativity. Using this strategy, we synthesized a number of switches and demonstrated its improvement of product titers and host growth controlling the complex deoxyviolacein biosynthesis pathway. Furthermore, we restored functions of a dysregulated multilayer NOR gate by integrating autorepression modules. Our work provides a blueprint for engineering host-adaptable synthetic systems.

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.

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.

CRISPR-Based Genetic Switches and Other Complex Circuits: Research and Application.

CRISPR-based enzymes have offered a unique capability to the design of genetic switches, with advantages in designability, modularity and orthogonality. CRISPR-based genetic switches operate on multiple levels of life, including transcription and translation. In both prokaryotic and eukaryotic cells, deactivated CRISPR endonuclease and endoribonuclease have served in genetic switches for activating or repressing gene expression, at both transcriptional and translational levels. With these genetic switches, more complex circuits have been assembled to achieve sophisticated functions including inducible switches, non-linear response and logical biocomputation. As more CRISPR enzymes continue to be excavated, CRISPR-based genetic switches will be used in a much wider range of applications.

Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E. coli.

The development of base editing (BE) technology has opened a new avenue for research studies in bacteriology, particularly for bacterial species in which the DNA double-strand breaks (DSBs) introduced by CRISPR/Cas system would lead to cell death. However, a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions, such as GC content. Herein, we developed a broad-spectrum DNase-inactive Cpf1 (dCpf1) variant from Francisella novicida (bsdFnCpf1) through directed evolution. The resulting optimized mutant showed a substantially expanded targeting range, including previously non-canonical protospacer-adjacent motifs (PAMs), especially the GC-rich PAMs. Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor (UGI) were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E. coli genome without compromising cell growth. We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1.

De novo design of an intercellular signaling toolbox for multi-channel cell-cell communication and biological computation.

Intercellular signaling is indispensable for single cells to form complex biological structures, such as biofilms, tissues and organs. The genetic tools available for engineering intercellular signaling, however, are quite limited. Here we exploit the chemical diversity of biological small molecules to de novo design a genetic toolbox for high-performance, multi-channel cell-cell communications and biological computations. By biosynthetic pathway design for signal molecules, rational engineering of sensing promoters and directed evolution of sensing transcription factors, we obtain six cell-cell signaling channels in bacteria with orthogonality far exceeding the conventional quorum sensing systems and successfully transfer some of them into yeast and human cells. For demonstration, they are applied in cell consortia to generate bacterial colony-patterns using up to four signaling channels simultaneously and to implement distributed bio-computation containing seven different strains as basic units. This intercellular signaling toolbox paves the way for engineering complex multicellularity including artificial ecosystems and smart tissues.

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.

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.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters.

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

A Direct RNA-to-RNA Replication System for Enhanced Gene Expression in Bacteria.

A long-standing objective of metabolic engineering has been to exogenously increase the expression of target genes. In this research, we proposed the permanent RNA replication system using DNA as a template to store genetic information in bacteria. We selected Qß phage as the RNA replication prototype and made many improvements to achieve target gene expression enhancement directly by increasing mRNA abundance. First, we identified the endogenous gene Rnc, the knockout of which significantly improved the RNA replication efficiency. Second, we elucidated the essential elements for RNA replication and optimized the system to make it more easily applicable. Combined with optimization of the host cell and the system itself, we developed a stable RNA-to-RNA replication tool to directly increase the abundance of the target mRNA and subsequently the target protein. Furthermore, it was proven efficient in enhancing the expression of specific proteins and was demonstrated to be applicable in metabolic engineering. Our system has the potential to be combined with any of the existing methods for increasing gene expression.

Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells.

With a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the DNase deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting. A screen of diversity-enriched PAM library was designed to investigate the PAM-dependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium. We found novel PAM patterns that elicited strong or medium gene repressions. Using a computational algorithm, we predicted regulatory outputs for all possible PAM sequences, which spanned a large dynamic range that could be leveraged for regulatory purposes. These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.