摘要
nsdA基因(negative regulator of streptomyces differentiation)是一个在链霉菌基因组中广泛存在的全局性负调控因子，天蓝色链霉菌的nsdA基因中断菌株能够提前且超量产生放线紫红素、钙依赖抗生素和次甲基霉素，同时产孢量也相应增多；阿维链霉菌NRRL8165的nsdA基因中断菌株的阿维菌素产量较出发菌株提高3-5倍。orfX基因是2003年在阿维链霉菌中发现的一个调控基因，研究发现含orfX基因的DNA片段转化变铅青链霉菌TK21后能显著提高放线紫红素和十一烷基灵菌红素的产量；用多拷贝质粒构建包含orfX基因的DNA片段的重组质粒，转化阿维链霉菌后显著提高了阿维菌素的产量。过去对这两个基因的研究工作均以原始菌株为材料，只是从理论上研究这两个基因的作用机制，未在工业菌株中应用。本研究的目的是研究nsdA基因中断和orfX基因整合表达对阿维链霉菌次级代谢的影响，探究它们在链霉菌工业育种中的作用。
本研究采用了PCR介导的基因置换技术构建nsdA基因中断菌株。合成一对长度分别为58，59nt的引物，其5’端的39nt序列分别与nsdA基因两侧同源，3’端则分别与安普霉素抗性标记盒(aac(3)Ⅳ+oriT)两侧序列一致。以该引物扩增的PCR产物电转化能表达λRed重组酶且含有目标质粒的Escherichia．coli菌株BW25113／pIR790／pBIB0501，获得了阳性重组质粒pBIB0502，将该质粒中的约4.5kbBglⅡ酶切片段插入基因置换载体pHL212，再接合转移至阿维链霉菌BIB9903，筛选得到表型为Apra~RThio~S的接合子BIB3009，PCR验证nsdA基因已被正确中断。YMS平板培养显示，BIB3009的产孢量较出发菌株明显减少，色素产量也明显降低。发酵分析以及HPLC分析显示，BIB3009的发酵液中菌丝量较出发菌株有所减少，阿维菌素产量大幅降低，为出发菌株的1／4-1／5，产量只有296μg／mL。
本研究根据GeneBank公布的阿维链霉菌基因组中预测的orfX基因序列(NP_828520.1)设计PCR引物P1、P2，以BIB9903总DNA为模板扩增orfX基因，以pIJ8600为出发质粒，构建了含红霉素抗性基因启动子(ermEp~*)和orfX结构基因的重组质粒pBIB827。通过接合转移将pBIB827导入阿维菌素的工业菌株BIB9903中，经PCR验证得到重组菌株BIB0827。YMS平板培养显示，BIB0827的产孢量较出发菌株明显增多，色素产量有所减少。发酵分析以及HPLC分析显示，BIB0827的发酵液中菌丝量较出发菌株有所增加，阿维菌素产量大幅提高，为出发菌株的2.5倍，产量达到3357μg／mL。传代稳定性实验证明，BIB0827传代10代后依然稳定，在工业生产上有较大的应用价值。
nsdA (negative regulator of Streptomyces differentiation) gene is a globally negativeregulator gene, which widely exists in Streptomyces. S. coelicolor nsdA null mutantsproduced more actinorhodin, CDA, methylenomycin and spores than wild type strain.Compared with the start strain of Streptomyces avermitilis NRRL8165, the level ofavermectin produced by S. avermitilis nsdA disruption strain increased 3-5 folds, orfXgene is a regulator gene discovered in the genome of S. avermitilis in 2003. The level ofactinorhodin and methylenomycin produced by S. lividans TK21 increased dramaticallywhen the DNA fragment containing orfX gene was transformed into it. When therecombinant plasmid containing the orfX gene, which was derived from multicopy vector,was transformed into S. avermitilis, the level of avermectin was elevated prominently. Inthe past, the study on the nsdA and orfX gene started with the wild type strains. Thefunction of the two genes was only investigated in theory and the possible industrialapplication was not carried out. The goal of this study is to test the effect of nsdA genedisruption and integrative expression of orfX gene on the secondary metabolism of S.avermitilis, and investigate the application of the two genes in the industrial breeding ofStreptomyces.
In this study, PCR-mediated gene replacement technique was applied in theconstruction nsdA gene disruption strain. Firstly, a pair of long (58,59nt) primers wereprepared, which have at 5'end 39nt matching the flanking sequence of nsdA, and the3'sequence matching the right or left end of the apramycin resistant cassettes(aac(3)Ⅳ+oriT). The linear DNA PCR-amplified by these primers waselectro-transformed to the strain BW25113/pIJ790/pBIB0501 which expressesλ-Redenzyme and contains target plasmid, then a positive recombined plasmid (pBIB0502) wasobtained. The 4.5kb fragment from pBIB0502 digested by BglⅡwas inserted intopHL212, and then introduced into S. avermitilis BIB9903 by conjugal transfer. AnApra~RThio~S isolate BIB3009 was obtained and nsdA disruption was confirmed by PCR.Compared with the start strain, BIB3009 produced fewer spores and less pigment onYMS plate. HPLC assays of the fermentation broth of BIB3009 indicated that the level ofavermectin decreased dramatically, and only 296μg/ml avermectin was produced, whichreached 1/4-1/5 of the yield of the start strain.
In this study, the PCR primers P1 and P2 were designed according to the predictedgene sequences of orfX gene in the S. avermitilis genome published in the GeneBank. orfX structural gene was amplified with genomic DNA of BIB9903 as template and P1and P2 as primers. Recombinant plasmid pBIB827, which contained the structural gene oforfX and ermEp~*, was derived from pIJ8600. The recombinant strain BIB0827 wasobtained by introducing pBIB827 into the industrial strain BIB9903 through intergenericgene transfer. Compared with the start strain, BIB0827 produced more spores and lesspigment on YMS plate. Fermentation assays indicated the biomass of the fermentationbroth of BIB0827 increased slightly when compared with the start strain. HPLC assaysshowed the level of avermectin produced by BIB0827 reached 3357μg/ml, 1.5-foldincreasing when compared with the start strain. The characteristic of high-level producingstrain BIB0827 was stable after ten rounds of non-selective growth, which could beapplied in the industrial production of avermectin.
引文
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