Gene editing enables rapid engineering of complex antibiotic assembly lines

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

A Concise Synthetic Strategy Towards the Novel Calcium-dependent Lipopeptide Antibiotic, Malacidin A and Analogues

Malacidin A is a novel calcium-dependent lipopeptide antibiotic with excellent activity against Gram-positive pathogens. Herein, a concise and robust synthetic route toward malacidin A is reported, employing 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis of a linear precursor, including late-stage incorporation of the lipid tail, followed by solution-phase cyclization. The versatility of this synthetic strategy was further demonstrated by synthesis of a diastereomeric variant of malacidin A and a small library of simplified analogues with variation of the lipid moiety.

Comparative Transcriptome Analysis Demonstrates the Positive Effect of the Cyclic AMP Receptor Protein Crp on Daptomycin Biosynthesis in Streptomyces roseosporus

Daptomycin, which is produced by Streptomyces roseosporus, has been characterized as a novel cyclic lipopeptide antibiotic that is effective against Gram-positive bacteria. The biosynthesis of daptomycin is regulated by various factors. In the present study, we demonstrated that the cyclic AMP receptor protein (Crp) plays an important role in producing daptomycin in the S. roseosporus industrial strain. We found that daptomycin production from the crp deletion strain decreased drastically, whereas production from the crp overexpression strain increased by 22.1%. Transcriptome and qPCR analyses showed that some genes related to the daptomycin biosynthetic gene cluster (dpt) and the pleiotropic regulator (adpA) were significantly upregulated. RNA-seq also shows Crp to be a multifunctional regulator that modulates primary metabolism and enhances precursor flux to secondary metabolite biosynthesis. These results provide guidance for the development and improvement of potential natural products.

The Use of Daptomycin in the Treatment of Persistent Coagulase-Negative Staphylococcal Sepsis in Premature Infants: A Case Series

OBJECTIVE Daptomycin is a lipopeptide antibiotic with rapid bactericidal activity against Gram-positive bacteria. Reports regarding the use of daptomycin in infants are still limited. Thus, the objective of this report is to describe the safety and efficacy of daptomycin in premature infants with persistent coagulase-negative staphylococci (CoNS) infection. METHODS This was a retrospective chart review of 10 premature infants with persistent CoNS infection who received daptomycin therapy between January 2018 and September 2019. Four patients had endocarditis and 1 had bacterial meningitis and infectious endocarditis. The other 5 patients had persistent CoNS bacteraemia only. RESULTS Daptomycin treatment was successful for 5 patients. The others died owing to multiple factors such as prematurity, sepsis, and chronic lung disease. Adverse drug reactions, including elevation of creatine phosphokinase and/or hepatotoxicity, were noted in 4 patients. CONCLUSIONS Large and randomized studies are necessary to ensure daptomycin's safety and efficacy for the treatment of infants with persistent sepsis caused by Gram-positive bacteria.

More Than a Pore: A Current Perspective on the In Vivo Mode of Action of the Lipopeptide Antibiotic Daptomycin

Daptomycin is a cyclic lipopeptide antibiotic, which was discovered in 1987 and entered the market in 2003. To date, it serves as last resort antibiotic to treat complicated skin infections, bacteremia, and right-sided endocarditis caused by Gram-positive pathogens, most prominently methicillin-resistant Staphylococcus aureus. Daptomycin was the last representative of a novel antibiotic class that was introduced to the clinic. It is also one of the few membrane-active compounds that can be applied systemically. While membrane-active antibiotics have long been limited to topical applications and were generally excluded from systemic drug development, they promise slower resistance development than many classical drugs that target single proteins. The success of daptomycin together with the emergence of more and more multi-resistant superbugs attracted renewed interest in this compound class. Studying daptomycin as a pioneering systemic membrane-active compound might help to pave the way for future membrane-targeting antibiotics. However, more than 30 years after its discovery, the exact mechanism of action of daptomycin is still debated. In particular, there is a prominent discrepancy between in vivo and in vitro studies. In this review, we discuss the current knowledge on the mechanism of daptomycin against Gram-positive bacteria and try to offer explanations for these conflicting observations.

Hypothesis: daptomycin permeabilizes membranes by forming self-assembled nanotubes

Daptomycin is the only lipopeptide antibiotic that is widely used in clinical practice. It was discovered by Eli Lilly and then studied and commercialized by Cubist Pharmaceuticals in 2003. Although this antibiotic has been used for 17 years, the debate over its mechanism of action is ongoing. In this paper, we discuss the different hypotheses on the mode of action of this antibiotic with a primary focus on the bacterial membrane permeabilization as the main mechanism of action. By comparing the experimental data on the oligomerization of daptomycin in membranes with properties of self-assembling cyclic peptides, we conclude that the structure of daptomycin oligomer should resemble the structures of peptide nanotubes that serve as ion channels in membranes.

Optimizing the Dosing Regimens of Daptomycin Based on the Susceptible Dose-Dependent Breakpoint against Vancomycin-Resistant Enterococci Infection

Daptomycin, a lipopeptide antibiotic, is one of the therapeutic options used for the treatment of vancomycin-resistant enterococci (VRE). Recently, the Clinical and Laboratory Standards Institute (CLSI) M100 30th edition has removed the susceptibility (S) breakpoint for Enterococcus faecium and replaced it with a susceptible dose-dependent (SDD) breakpoint of ≤4 μg/mL, with a suggested dosage of 8–12 mg/kg/day. Herein, we aimed to determine the minimum inhibitory concentration (MIC) values of daptomycin against clinical VRE isolates and to study the appropriate daptomycin dosing regimens among critically ill patients based on the new susceptibility CLSI breakpoint. The MIC determination of daptomycin was performed using E-test strips among clinical VRE strains isolated from patients at the Phramongkutklao Hospital. We used Monte Carlo simulation to calculate the probability of target attainment (PTA) and the cumulative fraction of response (CFR) of the ratio of the free area under the curve to MIC (fAUC0–24/MIC) > 27.4 and fAUC0–24/MIC > 20 for survival and microbiological response, respectively, at the first day and steady state. Further, we determined that the simulated daptomycin dosing regimen met the minimum concentration (Cmin) requirements for safety of being below 24.3 mg/L. All of the 48 VRE isolates were E. faecium strains, and the percentiles at the 50th and 90th MIC of daptomycin were 1 and 1.5 μg/mL, respectively. At MIC ≤ 2 μg/mL, a daptomycin dosage of 12 mg/kg/day achieved the PTA target of survival and microbiological response at the first 24 h time point and steady state. For a MIC of 4 μg/mL, none of the dosage regimens achieved the PTA target. For CFR, a dosage of 8–12 mg/kg/day could achieve the 90% CFR target at the first day and steady state. All dosing regimens had a low probability of Cmin being greater than 24.3 mg/L. In conclusion, the MIC of VRE against daptomycin is quite low, and loading and maintenance doses with 8 mg/kg/day were determined to be optimal and safe.