The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.
First Affiliated Hospital and Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou, China; Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou, China. Electronic address:
The study investigates how acylation, specifically crotonylation, regulates the production of daptomycin in the actinomycete Streptomyces roseosporus by modifying key metabolic enzymes.
Researchers found that crotonylation of the enzyme DptE, which starts the daptomycin biosynthesis process, limits its activity and identified the enzymes involved in adding and removing the crotonyl group.
By altering certain residues on DptE, the researchers enhanced its activity, significantly increasing daptomycin yield and suggesting potential improvements for industrial production through decrotonylation strategies.
Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S.
Lipopeptides are an important family of natural products, some of which are clinically used as antibiotics to treat multidrug-resistant pathogens. Although the lipid moieties play a crucial role in balancing antibacterial activity and hemolytic toxicity, modifying the lipid moieties has been challenging due to the complexity of the lipidation process in lipopeptide biosynthesis. Here, we show that the lipid profile can be altered by engineering both secondary and primary metabolisms, using daptomycin as an example.
Covering 2005-2024Daptomycin is a clinically important antibiotic that treats Gram-positive infections of skin and skin structure, bacteremia, and right-sided endocarditis, including those caused by methicillin-resistant (MRSA). Daptomycin is now generic, and many companies are involved in manufacturing and commercializing this life-saving medicine. There has been much recent interest in improving the daptomycin fermentation of by mutagenesis, metabolic engineering, and synthetic biology methods.
