ACS Synthetic Biology最新文献

Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. Bacillus subtilis is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in B. subtilis was established. The first module, driven by the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9 system. Another module responsible for expression control was systematically integrated into 28 discrete chromosomal loci and the impact of different genomic positions on gene expression was explored, resulting in a high-intensity integrated expression system. Furthermore, by modifying the LacI repressor factor for biological regulation, we achieved a strong expression intensity without the inducer addition. This system was successfully used to express phospholipase D and hyaluronic acid lyase, resulting in extracellular enzyme activities of 339.12 U/mL and 2.60 × 10⁴ U/mL, respectively. Additionally, by exclusively targeting the HA gene cluster for expression, a production yield of 6.86 g/L was achieved on a 5 L fermentation scale. The system eliminates the use of antibiotics and inducers, offering a controllable, efficient, and promising gene expression regulation tool in B. subtilis, enhancing its potential for biomanufacturing applications.

Since the description of the lac operon in 1961 by Jacob and Monod, transcriptional regulation in prokaryotes has been studied extensively and has led to the development of transcription factor-based biosensors. Due to the broad variety of detectable small molecules and their various applications across biotechnology, biosensor research and development have increased exponentially over the past decades. Throughout this period, key milestones in fundamental knowledge, synthetic biology, analytical tools, and computational learning have led to an immense expansion of the biosensor repertoire and its application portfolio. Over the years, biosensor engineering became a more multidisciplinary discipline, combining high-throughput analytical tools, DNA randomization strategies, forward engineering, and advanced protein engineering workflows. Despite these advances, many obstacles remain to fully unlock the potential of biosensor technology. This review analyzes the timeline of key milestones on fundamental research (1960s to 2000s) and engineering strategies (2000s onward), on both the DNA and protein level of biosensors. Moreover, insights into the future perspectives, remaining hurdles, and unexplored opportunities of this promising field are discussed.

The soil environment affected by plant roots and their exudates, termed the rhizosphere, significantly impacts crop health and is an attractive target for engineering desirable agricultural traits. Engineering microbes in the rhizosphere is one approach to improving crop yields that directly minimizes the number of genetic modifications made to plants. Soil microbes have the potential to assist with nutrient acquisition, heat tolerance, and drought response if they can persist in the rhizosphere in the correct numbers. Unfortunately, the mechanisms by which microbes adhere and persist on plant roots are poorly understood, limiting their application. This study examined the membrane proteome shift upon adherence to roots in two bacteria of interest, Klebsiella variicola and Pseudomonas putida. From this surface proteome data, we identified a novel membrane protein from a nonlaboratory isolate of P. putida that increases binding to maize roots using unlabeled proteomics. When this protein was moved from the environmental isolate to a common lab strain (P. putida KT2440), we observed increased binding capabilities of P. putida KT2440 to both abiotic mimic surfaces and maize roots. We observed a similar increased binding capability to maize roots when the protein was heterologously expressed in K. variicola and Stutzerimonas stutzeri. With the discovery of this novel binding protein, we outline a strategy for harnessing natural selection and wild isolates to build more persistent strains of bacteria for field applications and plant growth promotion.

Artepillin C is a diprenylated phenylpropanoid with various pharmacological benefits for human health. Its natural occurrence is limited to a few Asteraceae plants, such as Baccharis species, necessitating a stable supply through synthetic biology. In Saccharomyces cerevisiae, the utilization of aromatic substrates within the cell was limited, resulting in very low production of artepillin C. In this study, we used AcPT1, a p-coumaric acid (p-CA)-specific diprenyltransferase, in Komagataella phaffii to produce artepillin C. Detailed studies revealed that the critical bottleneck in K. phaffii was the supply of prenyl diphosphates, not phenylpropanoid flux. By enhancing the prenyl substrate pathway through overexpression of isopentenyl diphosphate isomerase and a truncated HMG-CoA reductase, we achieved a strong increase in artepillin C production. A major part of artepillin C was accumulated in yeast cells. One of the advantages of K. phaffii is its superior growth and ability to achieve high cell density cultivation compared to that of S. cerevisiae. Therefore, fed-batch cultivation with glycerol was performed. As a result, the dry cell weight (DCW) reached 61.0 g/L, and the intracellular amount of de novo produced artepillin C reached 187.3 μg/DCW. Analysis of intermediates revealed that the supply of p-CA constituted a bottleneck in artepillin C production in the engineered strain. By enhancing the p-CA supply, the intracellular accumulation of artepillin C reached 1200 μg/DCW even in batch cultivation. Moreover, the total intra- and extracellular amounts of artepillin C reached 12.5 mg/L, marking the highest de novo synthesis amount of artepillin C reported thus far, even under batch cultivation conditions.

Bacteria genetically engineered to execute defined therapeutic and diagnostic functions in physiological settings can be applied to colonize the human microbiome, providing in situ surveillance and conditional disease modulation. However, many engineered microbes can only respond to single-input environmental factors, limiting their tunability, precision, and effectiveness as living diagnostic and therapeutic systems. For engineering microbes to improve complex chronic disorders such as inflammatory bowel disease, the bacteria must respond to combinations of stimuli in the proper context and time. This work implements a previously characterized split activator AND logic gate in the probiotic Escherichia coli strain Nissle 1917 (EcN). Our system can respond to two input signals: the inflammatory biomarker tetrathionate and a second input signal, anhydrotetracycline (aTc), for manual control. We report 4-6 fold induction with a minimal leak when the two chemical signals are present. We model the AND gate dynamics using chemical reaction networks and tune parameters in silico to identify critical perturbations that affect our circuit's selectivity. Finally, we engineer the optimized AND gate to secrete a therapeutic anti-inflammatory cytokine IL-22 using the hemolysin secretion pathway in the probiotic E. coli strain. We used a germ-free transwell model of the human gut epithelium to show that our engineering bacteria produce similar host cytokine responses compared to recombinant cytokine. Our study presents a scalable workflow to engineer cytokine-secreting microbes driven by logical signal processing. It demonstrates the feasibility of IL-22 derived from probiotic EcN with minimal off-target effects in a gut epithelial context.

Bioproduction of chemicals by using engineered bacteria is promising for a circular economy but challenged the instability of the introduced plasmid by conventional methods. Here, we developed a two-plasmid INTEGRET system to reliably integrate the targeted gene into the Vibrio natriegens genome, making it a powerful strain for efficient and steady bioproduction without requiring antibiotic addition. The INTEGRET system allows for gene insertion at over 75% inserting efficiency and flexibly controllable gene dosages. Additionally, simultaneous gene insertion at four genomic sites was achieved at 54.3% success rate while maintaining stable inheritance of exogenous sequences across multiple generations. The engineered strain could efficiently synthesize PHB from the fermentation of diverse organic wastes, with an efficiency comparable to those with overexpressed plasmid. When the mixture of seawater and molasses was used as the feedstock, it achieved a high PHB yield of 39.41 wt %. An extended application of the INTEGRET system for imparting the riboflavin production ability to the bacterium was also demonstrated. Our work presents a reliable and efficient genomic editing tool to facilitate the development of sustainable and environmentally benign biological platforms for converting biomass wastes into valuable chemicals.

Competition among genes for limited transcriptional and translational resources impairs the functionality and modularity of synthetic gene circuits. Traditional control mechanisms, such as feedforward and negative feedback loops, have been proposed to alleviate these challenges, but they often focus on individual modules or inadvertently increase the burden on the system. In this study, we introduce three novel multimodule control strategies─local regulation, global regulation, and negatively competitive regulation (NCR)─that employ an antithetic regulatory mechanism to mitigate resource competition. Our systematic analysis reveals that while all three control mechanisms can alleviate resource competition to some extent, the NCR controller consistently outperforms both the global and local controllers. This superior performance stems from the unique architecture of the NCR controller, which is independent of specific parameter choices. Notably, the NCR controller not only facilitates the activation of less active modules through cross-activation mechanisms but also effectively utilizes the resource consumption within the controller itself. These findings emphasize the critical role of carefully designing the topology of multimodule controllers to ensure robust performance.

Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.

Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.

Controlling gene expression is useful for many applications, but current methods often require external user inputs, such as the addition of a drug. We present an alternative approach using cell-autonomous triggers based on RNA stem loop structures in the 3' untranslated regions (UTRs) of mRNA. These stem loops are targeted by the RNA binding proteins Regnase-1 and Roquin-1, allowing us to program stimulation-induced transgene regulation in primary human T cells. By incorporating engineered stem loops into the 3' UTRs of transgenes, we achieved transgene repression through Regnase-1 and Roquin-1 activity, dynamic upregulation upon stimulation, and orthogonal tunability. To demonstrate the utility of this system, we employed it to modulate payloads in CAR-T cells. Our findings highlight the potential of leveraging endogenous regulatory machinery in T cells for transgene regulation and suggest RNA structure as a valuable layer for regulatory modulation.