ACS Synthetic Biology最新文献

Historically, studying the development of brain and central nervous system (CNS) tissues has been challenging. Human pluripotent stem cell (hPSC) technology has allowed for the in vitro reconstitution of relevant, early cell trajectories by using small molecules and recombinant proteins to guide differentiation of cells toward relevant brain and CNS phenotypes. However, many of these protocols fail to recapitulate the cell-guided differentiation programs intrinsic to embryonic development, particularly the signaling centers that emerge within the neural tube during brain formation. Located on the ventral end of the neural tube, the floor plate acts as one such signaling center to pattern the dorsal/ventral axis by secreting the morphogen Sonic Hedgehog (SHH). Here, we present a method for cell-guided differentiation using the synthetic Notch (synNotch) receptor platform to regulate SHH production and subsequent cell fate specification. We show that the widely used configuration of the orthogonal synNotch ligand green fluorescent protein (GFP) mounted on a platelet-derived growth factor receptor-β transmembrane chassis does not allow for robust artificial signaling in synNotch-hPSCs ("receivers") cocultured with ligand-presenting hPSCs ("senders"). We discovered that refined designs of membrane-bound GFP-ligand allow for efficient receptor activation in hPSC receivers. A variant of this enhanced synNotch system drives the production of SHH in hPSC sender:hPSC receiver cocultures and gives rise to floor plate-like cell types seen during neural tube development. This revised synNotch platform has the potential to pattern hPSC differentiation programs in synthetic morphogenesis studies designed to uncover key paradigms of human CNS development.

Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the "Synthetic Gene Regulatory Network" (SGRN, pronounced "sojourn").

Optogenetics has emerged as a powerful tool for regulating cellular processes due to its noninvasive nature and precise spatiotemporal control. Far-red light (FRL) has increasingly been used in the optogenetic control of mammalian cells due to its low toxicity and high tissue penetration. However, robust and orthogonal FRL sensors are lacking in bacteria. Here, we established an orthogonal FRL sensor in Escherichia coli with a maximum dynamic range exceeding 230-fold based on the RfpA-RfpC-RfpB (RfpABC) signaling system that regulates the far-red light photoacclimation (FaRLiP) in cyanobacteria. We identified a conserved DNA motif in the promoter sequences of the Chl f synthase gene and other genes in the FaRLiP gene clusters, termed the far-red light-regulatory (FLR) motif, which enables the light-responsive activation of gene expression through its interaction with RfpB. Based on the FLR motif, we simplified the FLR-containing promoters and characterized their activation abilities and dynamic ranges, which can be utilized in different synthetic biology scenarios. Additionally, one or two FLR motifs are present at other loci within the FaRLiP gene cluster, providing further FRL-inducible promoter resources. The FRL sensor exhibits effective activation and suppression under low-intensity FRL and white light, respectively, and remains functional in darkness. In conclusion, this study advances the understanding of the regulatory mechanisms of FaRLiP in cyanobacteria and provides robust and orthogonal FRL sensors for synthetic biology applications.

Biofilms are ubiquitous and have many negative effects, for example, in infections or biocorrosion. Given the critical role of the second messenger cyclic di-GMP (c-di-GMP) in biofilm formation, targeting a reduction in intracellular concentrations of c-di-GMP is believed to be a key aspect in the development of biofilm mitigation strategies. To facilitate this effort, here, we developed a transcription factor (TF)-based biosensor that integrates the TF FleQ from Pseudomonas aeruginosa with a P_R-P_pel tandem promoter. The dynamic range of the biosensor was optimized by fine-tuning the TF expression. The biosensor exhibited broad compatibility and effectiveness in detecting decreases in c-di-GMP levels across various biofilm model organisms, including strains lacking FleQ or its homologues, such as Escherichia coli, Shewanella oneidensis, Comamonas testosteroni, and Acinetobacter baumannii, as well as P. aeruginosa containing FleQ. Additionally, we monitored c-di-GMP levels in biofilms formed by P. aeruginosa and S. oneidensis through a ratiometric, image-based quantification method. The methodology used the green fluorescence protein (GFP) as a reporter for c-di-GMP levels and 4',6-diamidino-2-phenylindole (DAPI) or the monomeric red fluorescence protein (mRFP) as the indicator for biofilm biomass. The GFP/DAPI or GFP/mRFP ratio gives effective c-di-GMP per unit of biomass. This TF-based biosensor provides an important tool to study c-di-GMP dynamics, which facilitates efforts in developing biofilm control strategies and understanding regulatory networks for biofilm development.

Immunoglobulins mediate their immune responses through interactions with Fc γ-receptors (FcγRs) on immune cells, triggering crucial responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). While enhancing these interactions can be beneficial, in certain therapeutic scenarios, such as cytokine or receptor blockade therapies, it is critical to reduce FcγR binding to avoid adverse immune reactions. This study aims to design negative mutations in the Fc region to reduce Fcγ receptor binding based on the residue interaction network analysis. The mutation sites of Fc were targeted through betweenness centrality analysis, and mutations were designed by focusing on hydrophobic to hydrophilic residue changes. The negative effect of the designed mutants on binding affinity was verified by previous reports and binding experiments. From this study, we identified a new Fc variant candidate (V263(B)D) that lacks a binding affinity for Fcγ receptors. This research highlights a strategic approach for designing Fc mutations that effectively reduce immune activation, which may be valuable in therapeutic contexts, where immune response moderation is crucial.

Spermidine finds broad applications across both the nutraceutical and biomedical sectors. In this study, key regulatory genes affecting spermidine synthesis and efficient integration sites were identified to construct a chassis strain for green and sustainable spermidine production. First, the expression of argJ was increased, and the protein SAM2 was mutated to promote the synthesis of spermidine. Second, positional effects were examined in Bacillus amyloliquefaciens. Concurrently, bioinformatics analysis was conducted to uncover transport proteins Blt, YvdR, and Mta, as well as other key genes tcyJ, yxeM, appC, yngA, and orf03307 that affect spermidine synthesis. Ultimately, strain PM13 was constructed through the iterative integration of key genes, achieving a spermidine titer of 396.92 mg/L, 10.34 times higher than strain PM1. Furthermore, xylose fed-batch fermentation increased spermidine titer to 1.69 g/L, setting a new shake flask production record. In conclusion, this study amassed genetic resources and developed an integrated strain for efficient, stable spermidine synthesis.

This study evaluates the performance of carbon monoxide dehydrogenase (codh)-embedded strains in bench-scale microbial electrochemical systems (MES) for CO₂ reduction to biofuels and biochemicals. CO₂ fermentation efficiency was evaluated by comparing the wild-type Clostridium acetobutylicum (Wild), a negative control E. coli strain lacking the codh gene (NC-BL21), and engineered E. coli strain (Eng) alone and with IPTG induction (Eng+IPTG). Four electrochemical systems were used, viz. Wild+E, NC-BL21+E, Eng+E, and Eng+IPTG+E, with a poised potential of -0.6 V applied to the working electrode. CO₂ and bicarbonate were supplemented to a total inorganic carbon (IC) concentration of 40 g/L, with a retention time of 60 h. The engineered strain demonstrated enhanced metabolic performance compared to the wild-type and negative control strains, yielding maximum formic acid (2.1 g/L) and acetic acid (9.3 g/L) under the Eng+IPTG+E condition. Solventogenesis also influenced positively in the same system with the ethanol yield of 3.9 g/L, substantially exceeding biochemicals yield observed in the wild-type strain (2.4 g/L, acetic acid). The engineered strains exhibited superior cumulative yields (0.40 g/g), enhanced CODH-mediated charge flux stability (60 vs 5 in the wild type), and upregulated expression of key genes in the Wood-Ljungdahl pathway (WLP). Bioelectrochemical performance analysis demonstrated elevated reductive catalytic currents, enhanced CO₂ reduction, and optimal charge transfer kinetics. This study highlights the synergistic potential of genetic engineering, specifically CODH overexpression, combined with electro-fermentation for enhanced biofuel and biochemical production from C1 gases.

Spider silk spidroins, nature's advanced polymers, have long hampered efficient in vitro production due to their considerable size, repetitive sequences, and aggregation-prone nature. This study harnesses the power of a cell-free protein synthesis (CFPS) system, presenting the first successful in vitro production and detailed characterization of recombinant spider silk major ampullate spidroins (MaSps) utilizing a reformulated and optimizedEscherichia coli based CFPS system. Through systematic optimization, including cell strain engineering via knockout generation, energy sources, crowding agents, and amino acid supplementation, we effectively addressed the specific challenges associated with recombinant spidroin biosynthesis, resulting in high yields of 0.61 mg/mL for MaSp1 (69 kDa) and 0.52 mg/mL for MaSp2 (73 kDa). The synthesized spidroins self-assembled into micelles, facilitating efficient purification compared to in vivo methods, and were further processed into prototype silk fiber products. The functional characterization demonstrated that the purified spidroins maintain essential natural properties, such as phase separation and fiber formation triggered by pH and ions. This tailored CFPS platform also facilitates versatile cosynthesis and serves as an accessible platform for studying the supramolecular coassembly and dynamic interactions among spidroins. This CFPS platform offers a viable alternative to conventional in vivo methods, facilitating innovative approaches for silk protein engineering and biomaterial development in a high-throughput, efficient manner.

Siderophore biosynthesis is severely inhibited by iron sufficiency, limiting microbial production of siderophores on a large industrial scale. Herein, we report novel iron-derepressed and robust production of the fungal siderophore fusarinine C (FsC) in Aureobasidium melanogenum, achieved by metabolic reprogramming coupling Raman-based single-cell sorting (RACS). First, we deciphered the mechanisms of iron repression on siderophore biosynthesis in this fungus. Guided by this, we constructed an iron-depressed chassis. In this chassis, we reprogrammed the metabolic pathways of FsC involving the modules of l-ornithine, mevalonate, and siderophore biosynthesis, accomplishing iron-depressed, robust, and sole production of FsC with an extracellular titer of 763 mg L^-1 under iron-sufficient conditions in stainless steel fermenters. However, intrinsic heterogenicity occurred in FsC bioproduction. To overcome this, we developed an approach based on flow-mode RACS to select and acquire high FsC-producing single cells from each fermentation batch to be used as the seed for the next batch, thereby persistently maintaining the robustness of the FsC titer over 818 mg L^-1 in successive batch fermentation. This study showcases the principle for overcoming iron repression and heterogenicity of siderophore bioproduction to fit large-scale industrialization.

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ (R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides, providing a streamlined method for addressing genetic engineering challenges in the yeast.