Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector

Abstract

Many strains of Amycolatopsis, such as Amycolatopsis orientalis, A. balhimycina, and A. mediterranei, are important antibiotic producers. Three indigenous plasmids, pMEA100, pMEA300, and pA387, found in this genus have been sequenced. However, only some vectors based on pA387 have been widely applied in Amycolatopsis research. An indigenous plasmid, pXL100, was isolated from the vancomycin producer A. orientalis HCCB10007. Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%. A 2830-bp fragment containing three ORFs has been identified as essential for replication in A. orientalis, but it has no significant similarity to any known replicons. A vector, pLYZW7-3, was constructed based on the pXL100 replicon and could be transferred into A. mediterranei and A. orientalis by electroporation or conjugation with high frequency. A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector, indicating that the vector is potentially useful in Amycolatopsis research.

Genetics and Genomics of the Genus Amycolatopsis

“…The occurrence of indigenous plasmids in this genus is also not very common. Until now, only six plasmids have been found in the genus Amycolatopsis [18] (Table 1). Till date, 30 Amycolatopsis genome projects have been completed, out of which six genomes have been completely sequenced, annotated and used extensively for research purposes (Table 2).…”

Role of two 5‐aminolevulinic acid biosynthetic pathways in heme and secondary metabolite biosynthesis in Amycolatopsis orientalis

“…All A. orientalis strains were grown on Gauze's synthetic agar medium (GM) for sporulation, cultivated at 28 °C with agitation at 220 rpm in shake‐flasks that contained seed medium for vegetative growth. Seed cultures were then transferred into shake‐flasks containing F1 , F2 , or F5 medium (60 g L −1 glycerol, 20 g L −1 yeast extract, 6 g L −1 KNO 3 , 0.2 g L −1 KH 2 PO 4 , 0.4 g L −1 MgCl 2 . 6H 2 O, 3 g L −1 CaCO 3 , pH 6.5), which were then incubated for 5 days to produce different products, that is, vancomycin, ECO‐0501, and coproporphyrin.…”

“…The homologous fragments of hemA2 were amplified using primer pairs HemA2‐UP‐F/HemA2‐UP‐R and HemA2‐DO‐F/HemA2‐DO‐R, and the fragments were inserted into the Hind III/ Xba I sites of pLYZL102 to produce pLYZLDA2. The resulting plasmids were introduced into strain LYGYQA by electroporation , and transformants were selected for on Bennett's medium (containing 50 mg L −1 apramycin). Isolated colonies were then electroporated with pLYZL104 to prompt a second crossover event.…”

“…The fermentation broth was extracted with methanol to yield a crude organic extract containing ECO‐0501, coproporphyrin, or vancomycin. HPLC methods for the separation of ECO‐0501 and vancomycin were conducted as previously described . Coproporphyrin production was assessed by HPLC using an Agilent Poroshell 120 EC‐C18 column (2.7 µm, 4.6 × 150 mm) at 390 nm with a flow rate 0.7 ml min −1 .…”

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

“…Until now, few plasmids have been described in Amycolatopsis species (22), and only four of them (pMEA100, pMEA300, pA387 and pXL100) have been further studied. However, only the replicative vectors derived from the endogenous plasmids pA387 and pXL100 have been shown to replicate into several Amycolatopsis species (12, 23).…”