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 B 1a and nemadectin, respectively. This work provides useful transporter tools and corresponding engineering strategy for Streptomyces .
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 ATPbinding 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 B 1a and nemadectin a 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.
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