ACS Synthetic Biology最新文献

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.

We herein developed an ultrasensitive and rapid strategy to identify genomic nucleic acids by integrating a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) into our recently developed isothermal technique, nicking and extension chain reaction system-based amplification (NESBA) reaction. In this technique, named CESBA, the NESBA reaction isothermally produces a large amount of RNA amplicons from the initial target genomic RNA (gRNA). The RNA amplicons bind to the crispr RNA (crRNA) and activate the collateral cleavage activity of Cas13a, which would then cleave the reporter probe nearby, consequently producing the final signals. Based on this design principle, we successfully detected SARS-CoV-2 gRNA as a model target very sensitively down to even a single copy (0.05 copies/μL) in both fluorescence- and lateral flow assay (LFA)-based modes with excellent specificity against other human coronaviruses (H-CoVs). We further validated the clinical applicability of CESBA by testing the 20 clinical samples with 100% clinical sensitivity and specificity. This work represents a potent and innovative strategy for the identification of genomic nucleic acids in molecular diagnostics, delivering exceptional levels of sensitivity.

Ectoine is an important natural secondary metabolite widely used in biomedical fields, novel cosmetics development, and the food industry. Due to the increasing market demand for ectoine, more cost-effective production methods are being explored. With the rapid development of synthetic biology and metabolic engineering technologies, the production of ectoine using traditional halophilic bacteria is gradually being replaced by higher-yielding and environmentally friendly nonhalophilic engineered strains. By introducing the ectoine synthesis pathway into model strains and optimizing the fermentation process through various metabolic regulations, high-level production of ectoine can be achieved. This review focuses on strategies for the microbial production of ectoine, including screening of wild strains, mutation breeding, and metabolic engineering of model strains, to elucidate the current research status and provide insights for the industrial production of ectoine.

The development of an engineered strain for efficient cytidine production holds significant value for both research and industrial applications. In this study, the pgi and edd genes were knocked out to reveal their roles involved in the regulation of efficient cytidine synthesis in Escherichia coli. The results showed that after 36 h of shaking flask fermentation, the pgi knockout strain E. coli NXBG-14 produced a cytidine concentration of 2.57 ± 0.04 g/L, and the pgi and edd double knockout strain E. coli NXBG-15 produced a cytidine titer of 2.68 ± 0.03 g/L, which represented enhancements of 1.68 and 1.75 times over the start strain, respectively. Transcriptome analysis revealed that the differentially expressed genes (DEGs) in the NXBG-14 strain were mainly enriched in the glycolytic pathway and the tricarboxylic acid (TCA) cycle. Additionally, ¹³C metabolic flow distribution indicated a significant increase in 6-phosphogluconate in the pentose phosphate pathway (PPP) for NXBG-15. These findings suggest that modifications of the pgi and edd genes redirect central carbon metabolism and promote cytidine accumulation.

In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.

We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq's ability to accurately detect variants under various experimental conditions. Finally, we show LevSeq's utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.

Galactose oxidase (GOase) is a versatile biocatalyst with a wide range of potential applications, ranging from synthetic chemistry to bioelectrochemical devices. Previous GOase engineering by directed evolution generated the M-RQW mutant, with unprecedented new-to-nature oxidation activity at the C6-OH group of glucose, and a mutational backbone that helped to unlock its promiscuity toward other molecules, including secondary alcohols. In the current study, we have used the M-RQW mutant as a starting point to engineer a set of GOases that are very thermostable and that are easily produced at high titers in yeast, enzymes with latent activities applicable to sustainable chemistry. To boost the generation of sequence and functional diversity, the directed evolution workflow incorporated one-shot computational mutagenesis by the PROSS algorithm and ancestral sequence reconstruction. This synergetic approach helped produce a rapid rise in functional expression by Pichia pastoris, achieving g/L production in a fed-batch bioreactor while the different GOases designed were resistant to pH and high temperature, with T₅₀ enhancements up to 27 °C over the parental M-RQW. These designs displayed latent activity against glucose and an array of secondary aromatic alcohols with different degrees of bulkiness, becoming a suitable point of departure for the future engineering of industrial GOases.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT7⁴) can increase the mutant frequency of the target gene by 1.12 × 10⁴-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT7⁴ system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT7⁴ to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.