2014.07.16【英译中】SCI 连载之二

小妮丫头 (流火) 路人甲
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发表于:2014-07-16 22:01 [只看楼主] [划词开启]

2014.07.16【英译中】SCI 连载之二



      The notion of “ribosome engineering” originally came from the finding, that a Streptomyces lividans strain with an altered ribosomal S12 protein that confers streptomycin resistance produced abundant quantities of the blue-pigmented antibiotic actinorhodin, although S. lividans normally does not produce antibiotics due to the dormancy of the antibiotic biosynthesis genes (Shima et al.1996). On the other hand, the bacterial alarmone ppGpp, produced on the ribosome, was found to bind to RNA polymerase (RNAP) (Artsimovitch et al.2004), eventually initiating the production of antibiotics (Bibb2005;Ochi 2007). This suggested that RNAP modification, by introducing a rifampicin resistance mutation, may mimic the ppGpp-bound form, activating the expression of biosynthetic gene clusters (Lai et al. 2002; Xu et al. 2002). Consequently, we have developed a method, termed ribosome engineering, to activate or enhance the production of secondary metabolites by targeting ribosomal protein S12, as well as other ribosomal proteins and translation factors, or RNAP, hypothesizing that bacterial gene expression may be increased dramatically by altering transcription and translation pathways. Ribosome engineering is characterized by its applicability to both strain improvement and silent gene activation to identify novel secondary metabolites. The fundamental mechanism by which ribosome engineering affects antibiotic production has been summarized in earlier reviews (Ochi et al. 2004; Ochi2007), as has the outline of this technology (Baltz 2011; Chiang et al.2011; Olano et al.2008; Xie et al.2009). Therefore, the present review highlights recent advances on this topic.

      “核糖体工程”的概念最早来自于Streptomyces lividans菌,对该菌种核糖体的S12蛋白进行处理,使其能够分泌大量蓝色抗生素——放线紫红素,让其具有链霉素抗性。但正常情况下,由于抗生素生物合成基因的沉睡状态,S. lividans是不会产生抗生素的。另一方面,发现一种细菌的信号素鸟苷四磷酸——多由核糖体产生——结合在RNA聚合酶上,然后开始进行抗生素的合成。这种现象说明,我们对RNAP进行利福平抗性突变修饰,可能会得到类似的鸟苷四磷酸结合体,进而激活生物合成基因簇的表达。由此,我们就得到了一种方法,也就是所说的核糖体工程,通过改变转录和翻译途径,在假设细菌基因表达能够得到显著增强的情况下,我们可以用靶核糖体蛋白S12激活或者提高次级代谢产物的产出,当然,还有其他核糖体蛋白和翻译因子或者RNAP。核糖体工程在挖掘新型次级代谢产物方面,同时具有菌种选育以及激活沉默基因的普遍适用性的特性。核糖体工程影响抗生素产量的基本原理在之前的综述已有说明,总的来说,是这项技术的一个梗概。因此,现在这篇综述的重点在于这项课题近几年的进展描述。

 

Impact on strain improvement

      Since many antibiotics, such as streptomycin, target the ribosome, ribosome mutants that confer antibiotic resistance may be obtained by simply selecting mutants on drug-containing plates, although some fraction of the mutants may be the ones affected in membrane permeability. Similarly, RNAP mutants may be obtained by growing bacteria on plates containing rifampicin that targets RNAP. This feasibility has yielded many successful examples of ribosome engineering, including the enhanced production of secondary metabolites and enzymes, as well as enhanced tolerance to toxic compounds such as 4-hydroxybenzoate (Table1). Ribosome engineering was effective in enhancing the yield of secondary metabolites in a wide range of structural classes, including polyketides, macrolides, aminoglycosides, and nucleosides. Importantly, the K88E and K88R mutations in rpsL(polypeptide amino acid numbering according to Streptomyces coelicolor), which encodes the ribosomal protein S12, and the H437Y and H437R in rpoB, which encodes the RNAP β subunit, were often effective (Table1). In all these mutant strains of S. coelicolor, overproduction of actinorhodin correlated with higher expression of act II-ORF4, a pathway-specific positive regulator of actinorhodin synthesis. Combinations of these drug-resistance mutations further enhanced bacterial productivity (Tamehiro et al. 2003; Tanaka et al. 2009a). For example, the introduction of eight different mutations enhanced actinorhodin production by S. Coelicolor 280-fold (Wang et al.2008) and the introduction of three mutations enhanced the production of the enzyme cycloisomaltooligosaccharide glucanotransferase by Paenibacillus agaridevorans 1,000-fold (Tanaka and Ochi, manuscript in preparation). Mutations in rpsL enhanced expression of the frr gene, which encodes ribosome recycling factor (Hosaka et al. 2006), and overexpression of frr in Streptomyces avermitilis increased avermectin production, even in an industrial strain (Li et al. 2010). Overexpression of frr may be a general method of boosting translation during the stationary phase, leading to reinforcement of secondary metabolism. The rif mutation S444F increased erythromycin production by Saccharopolyspora erythraea fourfold and metabolic changes induced by this mutation were analyzed in detail using DNA microarrays (Carata et al.2009).

      因为很多抗生素都是作用于核糖体,像链霉素就是,所以对于耐药性的核糖体突变型可以通过抗性平板进行简单筛选,当然,这其中不排除由于膜透性出现的假阳性。类似地,RNAP的突变体很可能存在于筛选RNAP的利福平抗性板子上的活菌当中。这种分析的可行性已经在核糖体工程试验的成功案例中多有验证,当然其中也包括对次级代谢产物及其酶的增产作用,对毒副作用化合物如4-羟苯酸酯的耐性增强。核糖体工程对大部分的次级代谢产物都有提高产量的作用,这其中包括多聚乙酰类、大环内酯类、氨基糖苷类以及核苷类。这其中,K88EK88R突变型(天蓝色链霉菌的多肽氨基酸酸值)中rpsL的基因,主要负责编码核糖体S12蛋白。H437YH437R突变型的rpoB的基因,负责编码RNAP的β大亚基,都是有效的作用位点。在S. coelicolor中的突变株中,放线紫红素的过量表达与act II-ORF4基因的高表达具有相关性。组合这些具有耐药性的突变株能够进一步强化细菌的生产能力。比如说,导入8种不同突变株可以强化S. Coelicolor中放线紫红素的产量280倍之多,引入3种突变株可以增强Paenibacillus agaridevorans的cycloisomaltooligosaccharide葡聚糖转移酶的表达1000倍。rpsL位点突变株能够强化编码核糖体循环因子frr基因的表达,而且frr基因在Streptomyces avermitilis中的过表达能够增加阿佛菌素的产量,即便是在工业菌株中也是如此。frr基因的过表达在静止培养期能够辅助翻译过程,促使次级代谢产物的生成,因此成为一种常用方法。Rif突变株S444F能够增加Saccharopolyspora erythraea中红霉素4倍产量,而且这种突变株的次级代谢变化已通过DNA微序列变化进行了详细分析。



最后编辑于:2014-07-17 12:44
分类: 英语

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