Optimized expression of oxazolomycins in engineered Streptomyces longshengensis and their activity evaluation

Abstract

Background: To cope with the growing number of severe diseases and intractable pathogens, drug innovation in both chemical structures and pharmacological efficiency has become an imperative global mission. Oxazolomycins are a unique family of polyketide-polypeptide antibiotics from Streptomyces with diverse functional groups in their structures, conferring them multifarious activities. But further development into clinical applications has been hindered for decades for many reasons. Among them, the yield improvement is a critical basis for activity evaluation and drug-like property optimization. This study aims to enhance the production of oxazolomycins in Streptomyces longshengensis through metabolic engineering and evaluate their bioactivity against clinically relevant pathogens.

Results: Co-transcriptional analyses suggested that two operons (the transcriptional unit from gene oxaG to oxaB, and that from gene oxaH to oxaQ) could be included in the oxazolomycin biosynthetic gene cluster (oxa BGC) of S. longshengensis. So a strategy was designed to replace the native promoter regions between oxaG and oxaH with constitutive promoters P_neo and P_kasO* following functional module evaluation. In the resultant strain (SL_OE), the production of oxazolomycin component Toxa5 was increased to 4-fold of that in the wild-type strain. Accordingly, the transcription of all related genes in oxa was clearly promoted. SL_OE was then subjected to sublethal dose of gentamicin to induce mutagenesis for optimizing the genetic background, generating a resistant mutant SL_ROE. With the introduction of transporter genes (ozmS and oxaA) into SL_ROE, 175 mg/L of Toxa5 was achieved, representing the highest yield in shake-flask fermentation to the best of our knowledge. Finally, the purified Toxa5 showed significant inhibition on the growth of clinically important Gram-negative pathogenic bacterium, Pseudomonas aeruginosa, and the biofilm formation of Bacillus subtilis. Intriguingly, an unprecedented antioxidant activity was also demonstrated.

Conclusions: An oxazolomycin high-producing system of S. longshengensis was established by employing genetic engineering strategies to facilitate the bioactivity exploitation. Oxazolomycin Toxa5 showed interesting inhibitory effects against multiple Gram-negative and -positive pathogens as well as antioxidant capacity, indicating its great potential in clinical applications. The findings provide an efficient strategy for the overproduction and activity evaluation of oxazolomycins.

Keywords: Streptomyces longshengensis; Bioactivity; Biosynthesis; Oxazolomycin.

Fig. 1

The biosynthesis of oxazolomycin and co-transcriptional analysis of the oxa gene cluster. (A) The organization of oxa BGC. The promoter regions are indicated with solid arrows. (B) The proposed biosynthetic pathway of oxazolomycin. In the biosynthetic pathway of oxazolomycin, malonyl-CoA and methoxymalonyl-ACP derived from primary metabolisms serve as the main precursors. They are sequentially loaded onto the corresponding polyketide synthesis modules to form a backbone, which is subsequently tailored by some enzymes to generate the final products [7, 19]. (C) Co-transcriptional analysis of genes in oxa

Fig. 2

Overexpression of key functional modules and their effect on oxazolomycin biosynthesis. (A) HPLC chromatogram of the fermentation broth from the wild-type strain. (B) Mass spectrum of oxazolomycin Toxa5. (C-E) Shematic drawing of the construction of plasmids pSET152::P_hrdB::ovmFGIH , pKC1139::P_hrdB::oxaB-G and pSET156::P_kasO*::oxaSA [14]. (F) HPLC analysis of oxazolomycin Toxa5 production in the engineered derivative strains. ns, the difference between these strains is not significant

Fig. 3

The promoter-replacement (PRE) strategy and its effect on oxazolomycin biosynthesis. (A) Construction of plasmid pKC1139::P_nk. (B) HPLC chromatograms of the fermentation products from WT and SL_OE. (C) HPLC analysis of oxazolomycin Toxa5 production in the wild-type strain and the engineered strain SL_OE. (D) Transcriptional analysis of oxa BGC in the wild-type strain and the engineered strain SL_OE.

Fig. 4

Effect of overexpressing the functional gene modules in SL_ROE on oxazolomycin production. (A) HPLC analysis of the fermentation broth of WT and SL_ROESA strains. The cross indicates the peak of Toxa5, while peaks I, II and III remained to be further analyzed. (B) The production of Toxa5 in different engineered strains analyzed by HPLC. (C) The growth curves of different strains. (D) The production curves of Toxa5 in different strains

Fig. 5

Analysis and preparation of oxazolomycins in high-yield producing strain SL_ROESA. (A) HPLC-HR-MS analyses of Toxa4 and Toxa6. (B) HPLC-HR-MS analyses of the proposed oxazolomycin A (OZM-A), B (OZM-B), and C (OZM-C). (C) The chemical structure of oxazolomycins. (D) The bioassays of different oxazolomycins using B. subtilis 1.1630 as the indicator strain. Toxa4, Toxa6 and oxazolomycin A-C refer to the peaks I, II and III in Fig. 4A, respectively.

Fig. 6

Evaluation of the antibacterial activity and biofilm formation of oxazolomycins. (A) Effects of different concentrations of Toxa5 on the growth of bacteria. (B) Effects of different concentrations of Toxa5 on biofilm formation. (C) Effects of different concentrations of Toxa5 on the biomass of B. subtilis in 24-well plate. (D) The biofilm of B. subtilis stained with crystal violet at different concentrations of Toxa5. (E) Representative SEM image of B. subtilis biofilm formation in the absence of Toxa5. (F) Representative SEM image of B. subtilis biofilm in the presence of 6.25 µg/mL of Toxa5. In C-F, B. subtilis 1.1849 was used for its higher sensitivity to Toxa5

Fig. 7

Antioxidant activity in vitro determined by ORAC assay and cytotoxicity of Toxa5. (A) Effect of different concentrations of Trolox on the fluorescence decay curves in ORAC assay. (B) The standard curve of trolox in ORAC assay. (C) Effect of different concentrations of Toxa5 on the fluorescence decay. (D) Effect of different concentrations of Toxa5 on the growth of A549 cell line