ACS Synthetic Biology最新文献

Due to its ability to utilize carbon dioxide, native intracellular accumulation of bioplastic precursors, and a high protein content, the bacterium Cupriavidus necator offers potential solutions for social problems tackled by modern biotechnology. Yet, engineering of high-performing chemolithotrophic production strains has so far been hindered by the lack of adequate genome editing methods. In this work we present the establishment of a lambda Red recombineering system for use in Cupriavidus necator H16. In combination with electroporation as DNA delivery system, it enables an efficient and fast gene deletion methodology utilizing either suicide plasmids or, for the first time, linear PCR product. The novel lambda Red system was validated for the modification of three different genomic loci and, as a proof-of-concept, ultimately utilized for stable genomic integration of Escherichia coli phytase gene appA into the phaC1 locus. A Cre/loxP system further enabled efficient marker recycling. The combination of a minimal transformation protocol with lambda Red recombineering and a Cre/loxP system offers a robust, freedom-to-operate synthetic biology tool in an increasingly important bacterial production host. This approach simplifies and accelerates genome engineering in C. necator and is expected to significantly enhance future strain development efforts.

Bacillus subtilis is a bacterial cell factory with outstanding protein secretion capabilities that has been deployed as a workhorse for the production of industrial enzymes for more than a century. Nevertheless, the production of other proteins with B. subtilis, such as antibody formats, has thus far been challenging due to specific requirements that relate to correct protein folding and disulfide bond formation upon export from the cytoplasm. In the present study, we explored the possibility of producing functional antibody formats, such as scFvs and scFabs, using the genome-reduced Midi- and MiniBacillus strain lineage. The applied workflow included selection of optimal chassis strains, appropriate expression vectors, signal peptides, growth media, and analytical methods to verify the functionality of the secreted antibody fragments. The production of scFv fragments was upscaled to the 1 L bioreactor level. As demonstrated for a human C-reactive protein-binding scFv antibody by mass spectrometry, biolayer interferometry, circular dichroism, free thiol cross-linking with N-ethylmaleimide, and nano-differential scanning fluorimetry, MidiBacillus strains can secrete fully functional, natively folded, disulfide-bonded, and thermostable antibody fragments. We therefore conclude that genome-reduced B. subtilis chassis strains are capable of secreting high-quality antibody fragments.

Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and PfAgo gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-PfAgo system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-PfAgo system can provide a new solution for the development of a portable, visual PEDV testing platform.

δ-Valerolactam (VL), as an organic compound, is an important precursor chemical for nylon and has a wide range of applications in organic synthesis, pharmaceutical synthesis, polymer materials, and other fields. This study introduces a novel biosynthetic method for producing VL in the engineered strain Escherichia coli BL21 through the reprogramming of polyketide synthases (PKS). Initially, an in vitro multienzyme system was constructed to verify the reliability of the VL synthesis pathway. Subsequently, an optimized biosynthetic pathway was established in E. coli, converting l-aspartate to VL with a yield of 3.66 mg/L in a 250 mL shake flask. Various engineering strategies were then implemented to enhance VL production, including substrate-enzyme affinity modification and multidimensional substrate optimization. These methods resulted in a 3.7-fold increase in VL yield, reaching 13.5 mg/L in shake flask cultures. Further scale-up in a 5 L fed-batch fermenter achieved a VL concentration of 76.2 mg/L. This research provides innovative insights into the optimization of VL production pathways and industrial-scale production.

The iGEM Grand Jamboree, hosted last year in Paris, France, represents a new way of sharing and communicating science. The global event for young synthetic biologists blends the serious biotech of a traditional conference with the spontaneous energy of a music festival, fostering a kind of World's Fair for the built-with-biology future. The broader biotech community should take inspiration from iGEM and seek to enrich more of our public communications and professional events with iGEM-like humanity, authenticity, and fun.

Zika virus infections remain severely underdiagnosed due to their initial mild clinical symptoms. However, recent outbreaks have revealed neurological complications in adults and severe deformities in newborns, emphasizing the critical need for accurate diagnosis. Lateral flow assays (LFAs) provide a rapid, cost-effective, and user-friendly method for antigen testing at point-of-care, bedside, or in home settings. LFAs utilizing nanobodies have multiple benefits over traditional antibody-based techniques, as nanobodies are much smaller, more stable, and simpler to manufacture. We introduce a nanobody-based LFA for the rapid identification of Zika virus antigens. Starting from two previously reported nanobodies recognizing the Zika nonstructural protein 1 (NS1), we evaluate periplasmic and cytosolic nanobody expression and test different purification tags and immobilization strategies. We quantify nanobody binding kinetics and validate their mutually noncompetitive binding. Avidity effects boost the capture of the tetrameric target protein by 3 orders of magnitude and point to a general strategy for higher sensitivity LFA sensing. The nanobody LFA detects Zika NS1 with a limit of detection ranging from 25 ng/mL in buffer to 1 ng/mL in urine. This nanobody-LFA has the potential to facilitate on-site and self-diagnosis, improve our understanding of Zika infection prevalence, and support public health initiatives in regions affected by Zika virus outbreaks.

Translation elongation is a multifaceted process that intricately links translational resource availability to the biophysical effects arising from the interaction of mRNA sequences, ribosomes, and nascent polypeptide chains. Optimizing (heterologous) gene expression via codon usage or tRNA preference alone may yield suboptimal outcomes. In this study, we present a comprehensive mechanistic model that accounts for the competition of tRNAs at the ribosomal A-site, internal Shine-Dalgarno sequence interactions, and the decelerating effects of positively charged peptide patches. Our model offers a holistic perspective on the effects of translational elongation, including growth rate-dependent variation in translational rates by 22 to 25% between slow- and fast-growing Escherichia coli cells. We emphasize that endogenous E. coli sequences typically adapt to these effects, particularly in highly expressed genes, where adaptation ensures efficient translation. Conversely, heterologous gene sequences from Saccharomyces cerevisiae are predicted to exhibit lower translational elongation rates by 14 to 70% compared to the homologous isoform. Simulated elongation profiles not only underscore potential sites for translation engineering but also suggest feasible synonymous codon exchanges. The implications of our model extend beyond mere codon usage adaptation and shed light on the key factors influencing translation efficiency (e.g., codons for positively charged amino acids reduced elongation rates by ∼6%). This study provides a nuanced understanding of the intricate dynamics governing translation in E. coli.

Brochosomes are proteinaceous nanostructures produced by leafhopper insects with superhydrophobic and antireflective properties. Unfortunately, the production and study of brochosome-based materials has been limited by poor understanding of their major constituent subunit proteins, known as brochosomins, as well as their sensitivity to redox conditions due to essential disulfide bonds. Here, we used cell-free gene expression (CFE) to achieve recombinant production and analysis of brochosomin proteins. Through the optimization of redox environment, reaction temperature, and disulfide bond isomerase concentration, we achieved soluble brochosomin yields of up to 341 ± 30 μg/mL. Analysis using dynamic light scattering and transmission electron microscopy revealed distinct aggregation patterns among cell-free mixtures with different expressed brochosomins. We anticipate that the CFE methods developed here will accelerate the ability to change the geometries and properties of natural and modified brochosomes, as well as facilitate the expression and structural analysis of other poorly understood protein complexes.

The sesquiterpene lactone parthenolide is a promising anticancer drug. Its biosynthesis via a microbial cell factory has been considered as a sustainable alternative to plant extraction. Herein, systematic metabolic engineering approaches, as well as the introduction of a novel noncanonical tricarboxylic acid (TCA) cycle, were employed to enhance the production of the key precursor germacrene A. By identifying two new dehydrogenases and controlling the expression of parthenolide synthase, we further achieved the elimination of byproducts and enhanced parthenolide production. A two-stage fermentation approach and in situ product extraction using macroreticular resin were further applied to relieve the nocuous effect of costunolide and parthenolide on the growth of yeast cell factories, ultimately achieving a titer of 549.7 mg/L for parthenolide and 972.7 mg/L for costunolide in a 10 L fermenter, which represents the highest reported titer obtained by microbial fermentation. The strategies should also contribute to the microbial cell factory-construction for other natural products exhibiting toxicity.

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds, such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including the ability to be assembled, freeze-dried, distributed, and then used at the point of need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid-handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semiautomated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride-sensing cell-free biosensors and demonstrate that they perform close to expected detection outcomes.