Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases-NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.

Valinomycin is a cyclodepsipeptide antibiotic with a broad spectrum of biological activities, such as antiviral, antitumor, and antifungal activities. However, the low yield of valinomycin often limits its applications in medicine, agriculture, and industry. In our previous report, Streptomyces sp. ZJUT-IFE-354 was identified as a high-yielding strain of valinomycin. In this study, Plackett-Burman design (PBD) and response surface methodology (RSM) were used to optimize components of medium. The optimal medium contained 31 g/L glucose, 22 g/L soybean meal, and 1.6 g/L K2HPO4·3H2O, which could generate 262.47 ± 4.28 mg/L of valinomycin. Then, the culture conditions were optimized by a one-factor-at-a-time (OFAT) approach. The optimal conditions for the strain included a seed age of 24 h, an inoculum size of 8% (v/v), an incubation temperature of 28 °C, an initial pH of 7.2, an elicitor of 0.1% Bacillus cereus feeding at 24 h cultivation, and the feeding of 0.6% L-valine at 36 h cultivation. The final valinomycin production increased to 457.23 ± 9.52 mg/L, which was the highest yield ever reported. It highlights that RSM and OFAT may be efficient methods to enhance valinomycin production by Streptomyces sp. ZJUT-IFE-354.

A novel strain with antifungal activity against Sclerotinia sclerotiorum was isolated from soil, and identified as Streptomyces sp. ZJUT-IFE-354 using morphological and 16S rDNA sequence analysis. The bioactive metabolite produced by strain ZJUT-IFE-354 was identified and characterized as valinomycin by spectroscopic and chemical methods. The yield of valinomycin was 191.26 mg/L from the culture of Streptomyces sp. ZJUT-IFE-354, which was the highest yield to our knowledge. The in vitro antifungal activity of valinomycin against S. sclerotiorum was investigated as 0.056 ± 0.012 (EC50) and 0.121 ± 0.023 μg/mL (EC95), respectively, which was approximately 10.696- and 30.960-fold more active than that of carbendazim. The results from scanning electron microscopy, cell membrane permeability, and D-sorbitol and ergosterol assay indicated that valinomycin exerted the antifungal activity probably by increasing permeability of fungal cell membrane, leading to mycelial electrolyte leakage, and eventually resulting in the death of S. sclerotiorum. Thus, valinomycin may be a promising antifungal agent to control S. sclerotiorum.
BACKGROUND: The online version contains supplementary material available at 10.1007/s13205-021-03055-5.

Fluorescence-based sensing in droplet microfluidics requires small sample volumes, allows for high-throughput assays, and does not suffer from photobleaching as each flowing sensor is only scanned one time. In this paper, we report a selective and sensitive fluorescence-based ion-sensing methodology in droplet microfluidics using a T-junction PDMS chip. The oil stream is doped with sensor ingredients including an ionophore, a cation exchanger, and a permanently cationic fluorophore as the optical reporter. Electrolyte cations from the aqueous sample are extracted into oil segments and displace the cationic dyes into aqueous droplets. Laser-induced fluorescence of the two immiscible phases is collected alternately, which is in clear contrast to most other ion-selective optode configurations such as nanoparticle suspensions that rely on mixed optical signals of two phases. The cation exchanger, tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, is found to dramatically enhance the dye emission in the nonpolar sensing oil by preventing ion-pairing interactions and aggregations of the dye molecules, providing new insights into the mechanism of cationic dye-based ion sensors. The high dye brightness allows us to use low concentrations of sensing chemicals (e.g., 10 μM) in the oil and attain high sensitivity for detection of ions in an equal volume of sample. Using valinomycin as the ionophore and methylene blue as the dye, K+ is detected with a response time of ∼11 s, a logarithmic linear range of 10-5 to 10-2 M, a 20-fold total fluorescence response, >1000-fold selectivity against other electrolyte cations, and negligible cross-sensitivity toward the sample pH. The K+ concentration in untreated and undiluted whole blood and sweat samples is successfully determined by this microfluidic sensing method without optical interference from the droplet sample to the sensing oil. Detection of other ionic analytes can be achieved using the corresponding ionophores.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the corresponding author. It has been found that Fig 2B contains manipulated components, and Fig 5A partially overlaps with Fig 6 of a published paper authored by Mirza Muhammad Faran Ashraf Baig, et, al., The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors, J. Pharm. Anal. 11 (2021) 174-182, https://doi.org/10.1016/j.jpha.2020.10.003. The corresponding author indicated that they cannot guarantee the integrity of the images in the manuscript, as well as the conclusions of the paper. As a result, the Editor-in-Chief has decided to retract the paper. The corresponding author deeply regrets the circumstances and apologizes to the scientific community for not having detected this prior to publication.

Valinomycin is a nonribosomal peptide that was discovered from Streptomyces in 1955. Over the past more than six decades, it has received continuous attention due to its special chemical structure and broad biological activities. Although many research papers have been published on valinomycin, there has not yet been a comprehensive review that summarizes the diverse studies ranging from structural characterization, biogenesis, and bioactivity to the identification of biosynthetic gene clusters and heterologous biosynthesis. In this review, we aim to provide an overview of valinomycin to address this gap, covering from 1955 to 2020. First, we introduce the chemical structure of valinomycin together with its chemical properties. Then, we summarize the broad spectrum of bioactivities of valinomycin. Finally, we describe the valinomycin biosynthetic gene cluster and reconstituted biosynthesis of valinomycin. With that, we discuss possible opportunities for the future research and development of valinomycin.

Human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been resulting in global epidemics with heavy morbidity and mortality. Unfortunately, there are currently no specific medicines that can better treat these coronaviruses. Drug repurposing is an effective and economical strategy for drug discovery from existing drugs, natural products, and synthetic compounds. In this review, the broad-spectrum antiviral activity of valinomycin (VAL), especially its activity against coronaviruses such as SARS-CoV, MERS-CoV, human coronavirus OC43 (HCoV-OC43), were summarized, it highlights that VAL has tremendous potential for use as a novel antiviral agent against SARS-CoV-2.

The aggregation of β-amyloid (Aβ) peptide in Alzheimer\'s disease (AD) is characterized by mitochondrial dysfunction and mitophagy impairment. Mitophagy is a homeostatic mechanism by which autophagy selectively eliminates damaged mitochondria. Valinomycin is a respiratory chain inhibitor that activates mitophagy via the PINK1/Parkin signaling pathway. However, the mechanism underlying the association between mitophagy and valinomycin in Aβ formation has not been explored. Here, we demonstrate that genetically modified (N2a/APP695swe) cells overexpressing a mutant amyloid precursor protein (APP) serve as an in vitro model of AD for studying mitophagy and ATP-related metabolomics. Our results prove that valinomycin induced a time-dependent increase in the mitophagy activation of N2a/APP695swe cells as indicated by increased levels of PINK1, Parkin, and LC3II as well as increased the colocalization of Parkin-Tom20 and fewer mitochondria (indicated by decreased Tom20 levels). Valinomycin significantly decreased Aβ1-42 and Aβ1-40 levels after 3 h of treatment. ATP levels and ATP-related metabolites were significantly increased at this time. Our findings suggest that the elimination of impaired mitochondria via valinomycin-induced mitophagy ameliorates AD by decreasing Aβ and improving ATP levels.

Ionophores have been integrated into various electrochemical and optical sensing platforms for the selective detection of ions. Previous ionophore-based optical sensors rely on a H+ chromoionophore as the signal transducer and consequently, suffered from a pH cross-response. pH independent methods were proposed very recently by utilizing the solvatochromic dyes or the exhaustive mode. Here, we report a pH independent sensing principle based on nanospheres containing ionophores. As the ion-exchange occurs, the signal transducer undergoes aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ), leading to a dramatic change in fluorescence intensity. The principle was evaluated on different ionophores including those selective for K+, Na+, Ca2+, and Pb2+. The nanospheres were also introduced into microfluidic chips and successfully applied for the determination of sodium and potassium ion concentrations in diluted blood serum and urine samples.

Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 μg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.