一种新的链霉菌表达载体启动子.doc

一种新的链霉菌表达载体启动子 作者：吴杭 张部昌 查向东 孔小卫 马清钧【摘要】 eryA基因直接控制着红霉素母环6-脱氧-红霉内酯B的合成，在红霉素生物合成过程中具有重要作用。本文克隆了eryA基因的启动子PeryA，以绿色荧光蛋白基因为报告基因，构建了大肠埃希菌-糖多孢红霉菌穿梭型质粒。PEG介导原生质体转化法将穿梭型质粒分别转入糖多孢红霉菌A226与变铅青链霉菌JT46，荧光显微镜检测发现，此启动子在两菌株中都具有功能。随后，以变铅青链霉菌JT46为宿主，对PeryA启动子区域进行了深入研究，结果发现该启动子的-35区并不是必需的，仅有-10区、长度为41bp的该启动子在链霉菌中仍具有功能。定点突变证明-10区对于该启动子是必不可少的。因此，41bp的该启动子片段可作为链霉菌的有效启动子，这是迄今为止所发现的最短的启动子之一，可用于构建新的链霉菌表达载体。 【关键词】 eryA基因； 启动子； 糖多孢红霉菌； 变铅青链霉菌； 增强型绿色荧光蛋白 ABSTRACT The eryA genes play a very important role in the biosynthesis of erythromycin macrolactone ring (6-deoxy-erythronolide B, 6-dEB). In this paper, an Escherichia coli-Saccharopolyspora erythraea shuttle vector containing the eryA promoter region was constructed using the enhanced green fluorescent protein (EGFP) gene as a reporter. The shuttle plasmid was transformed into Sac.erythraea A226 and Streptomyces lividans JT46 by PEG mediation, respectively. Fluorescence microscopy confirmed that the EGFP was expressed in both strains. Subsequently, the characteristics of the eryA promoter region were exclusively studied in the host strain S.lividans JT46, and the results showed that the predicted -35 region was not necessary for the promoter and the 41-bp promoter DNA fragment only containing predicted -10 region was still functioned in Streptomyces. Site-directed mutant demonstrated that the predicted -10 region was indispensable for the promoter. Thus, the 41-bp promoter segment can function as an effective promoter of Streptomyces expression vector, which is one of the shortest promoters hitherto found in Streptomyces and is useful for constructing new Streptomyces expression vectors. KEY WORDS eryA gene; Promoter; Saccharopolyspora erythraea; Streptomyces lividans; Enhanced green fluorescent protein (EGFP) Saccharopolyspora erythraea as a mycelium-forming actinomycete, is the major producer of erythromycin, a macrolide antibiotic that has an important application in clinic. Extensive genetic studies have provided some insights into the genes involved in the erythromycin biosynthesis1. It has been proposed that the biosynthesis of erythromycin might be separated into two stages1: (i) the synthesis of erythromycin macrolactone ring (6-deoxy-erythrolide B, 6-dEB) requiring one molecule of propionyl-CoA and six molecules of methylmalonyl-CoA; and (ii) the transformation of 6-dEB into erythromycin A in a series of reactions catalyzed by stereospecific hydroxylases, e.g. glycosyltransferases and methyltransferases. Similar to other secondary metabolic pathway genes25, the erythromycin biosynthetic genes exist in cluster on a Sac.erythraea chromosome. Nevertheless, there is no regulator associated with the ery genes of Sac.erythraea and no other gene in this organism has yet been shown to control erythromycin production1, which is obviously different from the antibiotic biosynthetic regulatory genes in most Streptomyces spp.6,7. In recent years, the yields of antibiotics have been greatly enhanced by overexpression or knockout of certain key genes and some new antibiotics have also been obtained by combinatorial biosynthesis811, which inspires us to be more interested in the expression system of Streptomyces sp. and Sac.erythraea. As an important element of gene expression, promoter is no doubt a key factor of understanding the gene expression systems. The central portion of erythromycin biosynthetic cluster contains the three eryA genes (eryAI, eryAII and eryAIII) encoding 6-deoxy-erythronolide B synthase (DEBS)12,13, which are co-transcribed under the control of eryA promoter. Reeve et al.14 have located the transcriptional initiation site of eryA promoter using S1 nuclease protection assay and predicted the -10 and -35 regions of the promoter by the alignment of 10 different promoters in ery gene cluster. However, a more detailed analysis of the promoter region has not yet been reported. In this study, an E.coli- Sac. erythraea shuttle vector was constructed using enhanced green fluorescent protein (EGFP) as a reporter in order to conveniently facilitate the investigation of the eryA promoter. As is well known, Streptomyces lividans has close kindred to Sac.erythraea and the species S.lividans has been the general host of Streptomyces in genetic manipulation because of its clear genetic background. Therefore, we chose S.lividans as the host strain to examine whether the eryA promoter was active in the strain. In addition, for further characterization of the eryA promoter region as a promoter of Streptomyces expression system, a series of novel report shuttle plasmids were constructed containing different eryA promoter DNA fragments and transformed into S.lividans. Through the fluorescence detection of the transformants, we confirmed a promoter that contains only -10 region of eryA promoter, from -41 to -1 relative to the start point of translation, was functional in the Streptomyces. The promoter has potential for constructing new Streptomyces expression vectors because of its very short length. 1 Materials and methods 1.1 Strains, plasmids and culture conditions Sac.erythraea A226 and S.lividans JT46 were gifts from Prof. Wang Yi-guang, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Sac.erythraea A226 was grown in tryptic soy broth (TSB; Difco, U.S.A.)15 for preparation of total DNA, and on R3M agar plate16 for the regeneration of protoplasts and the selection of transformants. Escherichia coli DH5 was used as a general cloning host and was grown and maintained according to the standard method17. The details of Sac.erythraea and S.lividans culture were referred to Kieser et al15. The vector pUC18 was used for genetic manipulation and cloning. Plasmids pEGFP-N1 (Clontech, U.S.A.), pUEKML18 and pWOR120-N18 were used for constructing reporter shuttle plasmids. 1.2 Cloning of the eryA promoter regions The genomic DNA of Sac.erythraea was isolated as described15. Based on the eryA gene sequences (GenBank accession no. M63676), four pairs of primers (Tab.1) were designed to acquire the promoter regions. Polymerase chain reaction (PCR) was used to amplify the PeryA and PeryA1 fragments and performed at 96 for 5 min, and then cycled 30 times at 94 for 1 min, 65 for 1 min, 72 for 2 min, followed by incubation at 72 for 5 min. The amplified products were recovered from an agarose gel, digested with AatII and NdeI, and ligated into the pUC18 to generate pUP and pUP1. However, through only denaturalization and annealing of forward and reverse primers, PeryA2 and PeryA3 fragments were obtained with AatII and NdeI adhesive ends and cloned into the pUC18 to create pUP2 and pUP3. 1.3 Construction of reporter shuttle plasmids To characterize the eryA promoter, pUPW-EGFP, a reporter shuttle plasmid, was constructed. The DNA fragment carrying multiply cloning sites (MCS) and fd transcription terminator was removed from pUEKML with NdeI and EcoRI and cloned into the same sites of pUP, yielding pUPKML. PCR was performed using plasmid pEGFP-N1 as a template, and the PCR product was cloned into the pUC18 to yield pUC-EGFP. The EGFP gene from pUC-EGFP was then subcloned into NdeI/HindIII- digested pUPKML to generate pUP-EGFP. Finally, a KpnI-digested and CIAP (calf intestinal alkaline phosphatase)- dephosphorylated pUP-EGFP was relocated to the same site of pWOR120-N, creating pUPW-EGFP. Other three reporter shuttle plasmids containing different eryA promoter DNA fragments were constructed as described above. 1.4 Transformation of Sac.erythraea and Streptomyces and microscopic analysis of transformants The preparation and transformation of Sac.erythraea and S.lividans protoplasts were described in literature 19 and 15, respectively. The plasmid pUPW-EGFP was introduced into Sac.erythraea A226 and S.lividans JT46 protoplasts by polyethylene glycol (PEG) mediation, thus creating A226 (pUPW-EGFP) and JT46 (pUPW-EGFP). The microscopy was carried out on a Zeiss Axiophot photomicroscope equipped with a fluorescence filter set for detecting EGFP of these transformants. Images were obtained with an Axiocam. The AxioVision software (AxioVision Version 4.1) was used to acquire images. 2 Results and discussion 2.1 Construction of reporter shuttle plasmid In order to investigate the promoter of erythromycin biosynthetic genes, the primers (Tab.1) were designed to amplify an eryA promoter region (PeryA) and the DNA fragment was cloned to yield pUP as shown in “Materials and methods”. Sequencing confirmed that the PeryA was entirely consistent with the corresponding sequence in reference13. In many organisms, the green fluorescent protein (GFP) of the jellyfish Aequorea victoria has proved to be a particularly useful and sensitive reporter20,21. The EGFP gene was a modified version of the GFP gene and was obtained from the eukaryotic expression plasmid pEGFP-N1. Previous works have demonstrated that the EGFP could be expressed in Sac.erythraea18. So we fused the PeryA to the promoterless EGFP gene to handily study the promoter activity. Thus, the reporter shuttle plasmid pUPW-EGFP was constructed by a series of subcloning as described in Fig.1. 2.2 Characteristic of the eryA promoter PermE (promoter of erythromycin resistance gene) is used widely for the expression of heterologous genes in Streptomyces and has comparatively strong, constitutive activity and relatively well characterized22. Moreover, the EGFP expression could be induced in Sac.erythraea under the control of the PermE from constructed Fig.1 Construction of report shuttle plasmid pUPW-EGFPplasmid pZM-EGFP18. Therefore, we took the A226 (pZM-EGFP) and JT46 (pZM-EGFP) transformants as positive controls. The plasmid pUPW-EGFP was transformed into Sac.erythraea A226 and S.lividans JT46 by PEG mediation, respectively. Then, the A226 (pUPW-EGFP) and JT46 (pUPW-EGFP) transformants were screened under the presence of thiostrepton. Myceliums of the four transformants all emitted green fluorescence. These results revealed that the EGFP was not only expressed in Sac.erythraea but also in Streptomyces, showing that the eryA promoter is active in both strains, namely, it seemed not to be genus-specific, and that the promoter was not controlled by pathway-specific regulators, which apparently differed from the promoters of most Streptomyces antibiotic biosynthetic genes23. Thus, it was imperative for us to further study its characteristics as a promoter of Streptomyces expression vector. 2.3 Determination of eryA promoter activity region in Streptomyces Evidences above have confirmed that the PeryA did work in Streptomyces. So it was necessary to characterize the minimal functional region of the eryA promoter in the host strain S.lividans. The PeryA1 fragment was cloned carrying the predicted -10 and -35 regions (Tab.1), and the plasmid pUP1W-EGFP was constructed. The transformant JT46 (pUP1W-EGFP) emitted green fluorescence. Furthermore, the plasmid pUP2W-EGFP was built containing only the predicted -10 region of the promoter. To our surprise, the EGFP expression in Streptomyces was still visible. To further test whether the predicted -10 region serves as an indispensable element of Streptomyces promoter, we mutated initial ATT of this region to CGG as shown in Tab.1. However, there was no EGFP expression in Streptomyces, meaning that the predicted -10 region was essential for the promoter. These results showed that the PeryA with only -10 region could function as an effective promoter of Streptomyces. Bibb et al22 pointed out that sequences corresponding to the consensus -35 region of the ermE promoter had greater variability than its “-10” sequence. By deletion mutation of Streptomyces coelicolor sapA gene promoter sequences, Hana24 found that even 18-bp promoter that extended from -8 to +10 relative to the transcriptional start point retained basal activity of the wild-type sapA promoter, and it was deduced that the adjoining vector DNA upstream of the 18-bp promoter was providing cis-acting sites for expression of the sapA gene. So we managed to align the upstream promoter sequences of the PeryA2 fragment and its adjoining pUC18 vector DNA. As shown in Fig.2, there was little similarity, which suggested that in accord with the results of Bibb22 and Hana24, the upstream promoter sequences of the predicted -10 region was also not conserved, predicating that it seemed not to be required for the eryA promoter in Streptomyces. Streptomycete genes have so far shown a wide diversity of promoter sequences and transcriptional patterns. The promoters fall into three basic categories at least25,26: (i) the promoters designated as E.coli, like Streptomyces promoters on the basis of similarity to the promoters recognized by the E.coli 70; (ii) promoters just similar to prokaryotic classical promoter 10 region; and (iii) promoters possessing no analogous sequences to -10 and -35 regions of prokaryotic typical promoters. In this study, we found that the eryA promoter may belong to the second type of Streptomyces promoters. To our best knowledge, this is the first report indicating that the predicted -35 region does not appear to be indispensable for the promoter in Streptomyces; moreover, the only 41-bp promoter DNA segment can work as an effective promoter of Streptomyces, which is very short. Thus, it will be very useful for building new Streptomyces expression vectors. Fig.2 Alignment of the upstream DNA sequences from translating site in pUP1W-EGFP and pUP2W-EGFP (The cloned DNA fragments carrying different eryA promoter regions are underlined【参考文献】 1 Mironov V A, Sergienko O V, Nastasiak I N, et al. Biogenesis and regulation of biosynthesis of erythromycins in Saccharopolyspora erythraea J. Appl Biochem Microbiol,2004,40(6):6132 Fernandez-Moreno M A, Martinez E, Boto L, et al. Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin J. J Biol Chem,1992,267(27):192783 Gandecha A R, Large S L, Cundliffe E. Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome J. Gene,1997,184(2):1974 Macneil D J, Occi J L, Gewain K M, et al. Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase J. Gene,1992,115(12):1195 Ruan X, Stassi D, Lax S A, et al. 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