ACS Synthetic Biology最新文献

To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using Bacillus subtilis was developed. This new method, named Seamed Express Assembly Method (SEAM)-combi-Ordered Gene Assembly in Bacillus subtilis (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform B. subtilis according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.

In mammals, Trimethylamine N-oxide (TMAO) is involved in various physiological processes, and is considered a biomarker for multiple diseases. As a natural molecule found in marine organisms, TMAO is also an important indicator of seafood freshness. In this study, a TMAO biosensor was developed in Escherichia coli harnessing TorRST two-component system. By using a cascade amplification circuit based on HrpRS-P_hrpL, the biosensor's dynamic range was increased from 4.1- to 10.3-fold. By optimizing the affinity between the regulatory protein TorR and DNA binding sites in promoters, the concentration for 50% of maximal effect (EC₅₀) value was reduced from 1008 to 141 μM. The biosensor was successfully used for aquatic sample detection. By introducing an exogenous TMAO degradation pathway into E. coli Nissle 1917, a probiotic chassis capable of TMAO detection, transportation, and degradation was constructed, providing an effective tool for rapid detection of TMAO and prevention of multiple diseases.

The COVID-19 pandemic has highlighted the critical need for pathogen detection methods that offer both low detection limits and rapid results. Despite advancements in simplifying and enhancing nucleic acid amplification techniques, immunochemical methods remain the preferred methods for mass testing. These methods eliminate the need for specialized laboratories and highly skilled personnel, making home testing feasible. Here, we developed nanobody-based lateral flow assays (LFAs) for the rapid detection of SARS-CoV-2 and MERS-CoV in single and dual formats as point-of-care diagnostic tools. The developed LFAs are highly sensitive and successfully detected analytes at clinically relevant diagnostic cutoff values. Additionally, our results confirmed that the LFAs have a long shelf life and can be produced cost-effectively and with ease.

Cannabichromene (CBC), a valuable but extremely low-abundance component of cannabinoids in Cannabis sativa L., is known for its ability to promote neurogenesis. The scarcity of CBC in natural C. sativa is primarily attributed to the inefficiency of the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4 phosphate (DOXP/MEP) and fatty acid metabolism pathways, along with the limited competitive advantage of cannabichromenic acid synthetase (CBCAS) compared to other cannabinoid synthases. In this study, we constructed Saccharomyces cerevisiae capable of biosynthesizing cannabichromenic acid (CBCA) from glucose and olivetolic acid. First, we enhanced the supply of the precursor isopentenyl diphosphate/dimethylallyl diphosphate by introducing a two-step isopentenol utilization pathway (IUP). Additionally, we increased the CBCA titer by co-overexpressing endoplasmic reticulum auxiliary protein genes. Moreover, we improved the selectivity and catalytic activity of CBCAS through rational design. By localizing the IUP to peroxisomes, geranylgeranyl pyrophosphate and CBCA titers were further increased by 1.6-fold and 28%, respectively. Notably, the yeast strain synthesized CBCA at a rate 25.8% higher than that of C. sativa. Our findings suggest that microbial synthesis offers a promising alternative to traditional C. sativa for sustainable CBCA production.

Engineered living materials (ELMs) integrate aspects of material science and biology into a unique platform, leading to materials and devices with features of life. Among those, ELMs containing microalgae have received increased attention due to the many benefits photosynthetic organisms provide. Due to their relatively recent occurrence, photosynthetic ELMs still face many challenges related to reliability, lifetime, scalability, and more, often based on the complicated crosstalk of cellular, material-based, and environmental variables in time. This Viewpoint aims to summarize potential avenues for improving ELMs, beginning with an emphasis on understanding the cell's perspective and the potential stresses imposed on them due to recurring flaws in many current ELMs. Potential solutions and their ease of implementation will be discussed, ranging from choice of organism, adjustments to the ELM design, to various genetic modification tools, so as to achieve ELMs with longer lifetime and improved functionality.

Ectoine, a major compatible solute in halophilic micro-organisms, shows great potential in cosmetics and pharmaceuticals areas owing to its water-binding properties and capability to prevent oxidative damage. In this study, the ectABC gene cluster responsible for the ectoine synthesis originated from halophilic bacterium Halomonas venusta was first assembled into Escherichia coli. Subsequently, the crr gene in PTS was knocked out to further drive the metabolic flux from phosphoenolpyruvate to oxaloacetate, resulting in 1.27 g/L of ectoine. Then, the rate-limiting enzyme LysC in the ectoine synthesis pathway was identified and modified. The recombinant E. coli with the further overexpression of feedback-insensitive mutant EclysC* increased the ectoine titer to 2.51 g/L with a yield of 0.37 g/g in shake flasks. After the medium optimization including the carbon and nitrogen source, sodium chloride, and magnesium sulfate concentration, the ectoine titer was improved to 4.55 g/L. 115.15 g/L of ectoine with a yield of 0.23 g/g was obtained in the 5.0 L bioreactor through the optimization of substrate feeding and IPTG supplementation in the fed-batch fermentation. To achieve the cost-effective production of ectoine, lignocellulosic hydrolysate from wheat straw was adopted. 134.08 g/L of ectoine with a yield of 0.33 g/g sugar and a productivity of 3.7 g/L/h was finally produced, representing a relatively high level of ectoine production from renewable resources compared to other studies. This study provides valuable insights into a cost-effective and efficient method for industrial-scale ectoine production.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharides (HMOs). 2'-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting the growth of intestinal probiotics. The microbial cell factory technique has shown promise for the massive production of 2'-FL. Here, we aimed to construct a recombinant E. coli BL21(DE3) strain for the hyperproduction of 2'-FL. Initially, multicopy genomic integration and expression of the lactose permease gene lacY reduced the formation of byproducts. Furthermore, a more efficient Shine-Dalgarno sequence was used to replace the wild-type sequence in the manC-manB and gmd-wcaG gene clusters, which significantly increased the 2'-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the pgi gene. This further improved 2'-FL synthesis when glycerol was used as the sole carbon source. Finally, a new α-1,2-fucosyltransferase was identified in Neisseria sp., which exhibited a higher capacity for 2'-FL production. Fed-batch fermentation produced 141.27 g/L 2'-FL in 45 h with a productivity of 3.14 g/L × h. This productivity rate achieved the highest recorded 2'-FL levels, indicating the potential of engineered E. coli BL21 (DE3) strains for use in the industrial production of 2'-FL.

Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted in vivo evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in Aspergillus nidulans NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated in vivo evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.

Cell-free synthetic biology incorporates purified components and/or crude cell extracts to carry out metabolic and genetic programs. While protein synthesis has historically been the primary focus, more metabolism researchers are now turning toward cell-free systems either to prototype pathways for cellular implementation or to design new-to-nature reaction networks that incorporate environmentally relevant substrates or new energy sources. The ability to design, build, and test enzyme combinations in vitro has accelerated efforts to understand metabolic bottlenecks and engineer high-yielding pathways. However, only a small fraction of metabolic possibilities has been explored in cell-free systems, and extracts from model organisms remain the most common starting points. Expanding the scope of cell-free metabolism to include extracts from new organisms, alternative metabolic pathways, and non-natural chemistries will enhance our ability to understand and engineer bio-based chemical conversions.

Maintaining the efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse transcriptase ACTivity crispR (REACTR) for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to the synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.