Characterization of a LAL-type regulator NemR in nemadectin biosynthesis and its application for increasing nemadectin production in Streptomyces cyaneogriseus

Nemadectin, a macrocyclic lactone antibiotic, is produced by Streptomyces cyaneogriseus ssp. noncyanogenus. A methoxime derivative of nemadectin, moxdectin, has been widely used to control insect and helminth in animal health. Despite the importance of nemadectin, little attention has been paid to the regulation of nemadectin biosynthesis, which has hindered efforts to improve nemadectin production via genetic manipulation of regulatory genes. Here, we characterize the function of nemR, the cluster-situated regulatory gene encoding a LAL-family transcriptional regulator, in the nemadectin biosynthesis gene cluster of S. cyaneogriseus ssp. noncyanogenus NMWT1. NemR is shown to be essential for nemadectin production and found to directly activate the transcription of nemA1-1/A1-2/A2, nemC and nemA4/A3/E/D operons, but indirectly activate that of nemG and nemF. A highly conserved sequence 5′-TGGGGTGKATAGGGGGTA-3′(K=T/G) is verified to be essential for NemR binding.Moreover, four novel targets of NemR, including genes encoding an SsgA-like protein(TU94_12730), a methylmalonyl-CoA mutase(TU94_19950), a thioesterase of oligomycin biosynthesis(TU94_22425) and a MFS family transporter(TU94_24835)are identified. Overexpression of nemR significantly increased nemadectin production by 79.9%, in comparison with NMWT1,suggesting that nemR plays an important role in the nemadectin biosynthesis.

Transcriptome-guided identification of a four-component system, SbrH1-R, that modulates milbemycin biosynthesis by influencing gene cluster expression, precursor supply, and antibiotic efflux

Streptomyces can produce numerous antibiotics and many other bioactive compounds.Recently,the molecular mechanisms of transcriptional regulators in control of antibiotic production by influencing the expression of biosynthetic gene clusters(BGCs)have been extensively studied.However,for regulators that affect both antibiotic production and cell growth,the way to influence antibiotic production may be diverse,but related studies are limited.Here,based on time-course transcriptome analysis,a four-component system,SbrH1-R,consisting of the two-component system SbrKR(SBI_03479/3478)and two hypothetical proteins SbrH1(SBI_03481)and SbrH2(SBI_03480)potentially related with the biosynthesis of milbemycins was identified in Streptomyces bingchenggensis BC-101-4.Deletion of sbrH1-R resulted in weakened cell growth but a 110%increase of milbemycin production compared with that in BC-101-4.Comparative transcriptome analyses of the sbrH1-R mutant and BC-101-4 revealed that SbrH1-R not only indirectly represses milbemycin BGC expression,but also inhibits milbemycin production by modulating expression levels of genes related to precursor supply and antibiotic efflux.Further genetic experiments identified several new targets,including five precursor supply-associated reactions/pathways(e.g.,the reaction from pyruvate to acetyl-CoA,the reaction from acetyl-CoA to citrate,the fatty acidβ-oxidation process,and the branched chain amino acid and phenylalanine acid degradation pathways)and a milbemycin exporter system(MilEX2)that can be engineered for milbemycin overproduction.These results shed new light on the understanding of regulation of milbemycin biosynthesis and provide useful targets for future metabolic engineering of the native host to improve milbemycin production.

Mining and fine-tuning sugar uptake system for titer improvement of milbemycins in Streptomyces bingchenggensis

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 finetuned 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.