LL-F 28249gamma

Abamectin (ABA) constitutes a big commodity for pharmaceutical companies because it generates about one billion dollar annual sale. Avermectins (AVMs) and their naturally occurring analogues, milbemycins (MILs), meilingmycins (MEIs), ivermectin (IVE), abamectin (ABA), and nemadectin (NEM), represent one of the most developed antiparasitic agents. Abamectin is a mixture of avermectin B1a and avermectin B1b. The production of abamectin by Streptomyces avermitilis is a complicated process and separation of two fractions is quite difficult; commercial product contains more than 80% of Bla and less than 20% of B1b components. The main goal of the study was the identification and optimization of fermentation conditions to raise the production of abamectin from Egyptian S. avermitilis. The qualitative and quantitative analysis of avermectins was carried out by thin layer chromatography (TLC) and 6538 Q-TOF with Agilent 1290 UHPLC. The process of identification was carried out by using production medium containing 30 g/L corn starch, and 0.725 g/L CaCO3, pH 7, 8% inoculum size and incubated at 32.5 °C. The enhancement of the production of abamectin is a big challenge with commercial and industrial importance, as its output is insufficient for human consumption.

Exporter engineering is a promising strategy to construct high-yield Streptomyces for natural product pharmaceuticals in industrial biotechnology. However, available exporters are scarce, due to the limited knowledge of bacterial transporters. Here, we built a workflow for exporter mining and devised a tunable plug-and-play exporter (TuPPE) module to improve the production of macrolide biopesticides in Streptomyces. Combining genome analyses and experimental confirmations, we found three ATP-binding cassette transporters that contribute to milbemycin production in Streptomyces bingchenggensis. We then optimized the expression level of target exporters for milbemycin titer optimization by designing a TuPPE module with replaceable promoters and ribosome binding sites. Finally, broader applications of the TuPPE module were implemented in industrial S. bingchenggensis BC04, Streptomyces avermitilis NEAU12 and Streptomyces cyaneogriseus NMWT1, which led to optimal titer improvement of milbemycin A3/A4, avermectin B1a and nemadectin α by 24.2%, 53.0% and 41.0%, respectively. Our work provides useful exporters and a convenient TuPPE module for titer improvement of macrolide biopesticides in Streptomyces. More importantly, the feasible exporter mining workflow developed here might shed light on widespread applications of exporter engineering in Streptomyces to boost the production of other secondary metabolites.

Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase, when antibiotics are extensively produced. Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macrolide biopesticides, while the low yield and titer impede their broad applications in agricultural field. Considering that inadequate sugar uptake generally hinders titer improvement of desired products, we mined the underlying sugar uptake systems and fine-tuned their expression in this work. First, we screened the candidates at both genomic and transcriptomic level in S. bingchenggensis. Then, two ATP-binding cassette transporters named TP2 and TP5 were characterized to improve milbemycin titer and yield significantly. Next, the appropriate native temporal promoters were selected and used to tune the expression of TP2 and TP5, resulting in a maximal milbemycin A3/A4 titer increase by 36.9% to 3321 mg/L. Finally, TP2 and TP5 were broadly fine-tuned in another two macrolide biopesticide producers Streptomyces avermitilis and Streptomyces cyaneogriseus, leading to a maximal titer improvement of 34.1% and 52.6% for avermectin B1a and nemadectin, respectively. This work provides useful transporter tools and corresponding engineering strategy for Streptomyces.