2014.07.18【英译中】SCI 连载之三

小妮丫头 (流火) 路人甲
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发表于:2014-07-18 10:30 [只看楼主] [划词开启]

2014.07.18【英译中】SCI 连载之三


Construction of an amenable host organism for heterologous gene expression is a current goal for enhancing yield and activating cryptic gene clusters (Baltz2010; Komatsu et al.2010). Introduction of the rpsL K88R mutation into Streptomyces fradiae resulted in the efficient heterologous expression of secondary metabolite genes (Alexander et al.2010). Moreover, introduction of the rpsL K88E and rpoB S433L mutations into S. coelicolor enhanced the production of chloramphenicol and congocidine 40- and 30-fold, respectively (Gomez-Escribano and Bibb2011), and mutations conferring resistance to lincomycin and kanamycin enhanced violacein production in Escherichia coli 41-fold (Ahmetagic and Pemberton2011). Although the present review does not focus on the host organism system for heterologous expression of secondary metabolite gene clusters, the most updated review can be seen elsewhere (Komatsu et al.2013).

构建一种能够适合异源基因表达的合适宿主,是目前如何增产以及激活隐藏基因簇的一个亟待解决的目标。在Streptomyces fradiae中导入rpsL 基因K88R型突变,促使次级代谢产物基因得到有效的异源表达。此外,在S. coelicolor导入rpsL 基因K88E型突变和rpoB 基因S433L型突变,使得该菌氯霉素产量达到40倍,刚果杀菌素达到30倍之多,并且,对林可霉素和卡那霉素有抗性的突变株能够增强大肠杆菌中紫色杆菌素的产量达到41倍。虽然这篇综述的重点不在次级代谢基因簇的异源表达生物体系统研究上,这方面的文献可以在其他地方找到。

The rsmG gene encodes a 16S rRNA methyltransferase, which methylates position G527 of the 530 loop of 16S rRNA (Okamoto et al. 2007). Mutations of this gene in Streptomyce strains confer low-level resistance to streptomycin and cause preferential overexpression of the metK gene (at least in S.coelicolor), which encodes the enzyme Sadenosylmethionine (SAM) synthetase, eventually leading to an increase in intracellular SAM level (Nishimura et al. 2007). This increase, together with enhanced protein synthesis during the late growth phase, results in overproduction of antibiotic (Kim et al. 2003; Okamoto et al. 2003). Addition of SAM to the medium or propagation of metK with multicopy plasmids also results in antibiotic overproduction by various Streptomyces spp. and other actinomycetes (Huh et al.2004; Maharjan et al.2008, 2012; Oh et al. 2010; Paudel et al. 2011; Saito et al. 2003; Shin et al. 2007; Sun et al.2012; Wang et al. 2007; Zhao et al. 2006, 2010). Spontaneous rsmG mutations arise at a high frequency (10−4 10−6 ) (Nishimura et al. 2007; Okamoto et al. 2007). rsmG mutation (and also ksgA mutation[Ochietal.2009]) harbors several mysterious features (Nodwell2007) and is effective for not only enhanced antibiotic production but also activation of cryptic secondary metabolite biosynthetic genes (Tanaka et al. 2009b). When constructing strains combining low-level (rsmG) and high-level (rpsL) streptomycin resistance mutations, the mutations should be introduced in that order (Tanaka et al.2009a).

rsmG基因负责编码16S rRNA 的甲基转移酶。这种酶能在16S rRNA530环的G527位点进行甲基化作用。Streptomyce中的这个基因的突变株对链霉素的耐药性比较差,而且会使编码S-腺蛋氨酸合成酶的metK基因优先过量表达,最后增强了胞内的S-腺蛋氨酸水平。这种增长水平,常在生长期晚期增强蛋白的合成,结果使得抗生素的过表达。培养基中添加S-腺蛋氨酸或者含metK基因的多拷贝质粒也会使Streptomyces属中很多种以及其他放线菌中的抗生素过量表达。rsmG基因的自发突变通常突变频率较高,在10−410−6左右。rsmG基因突变株常含有一些未知的特性,它不仅能够增强抗生素产量,还能激活潜在的次级代谢产物合成酶基因。在结合低水平(rsmG)和高水平(rpsL)耐药性突变株构建中,突变体的导入需要有前后顺序。

 

Impact on silent gene activation

沉默基因激活的影响因素

Promising approaches for the activation of cryptic biosynthetic gene clusters in Streptomyces species include ribosome engineering; the addition of N-acetylglucosamine to the medium or deletion of the dasR gene, which encodes an N-acetylglucosamine-responsive regulatory protein, the constitutive overexpression of a pathway-specific large ATP-binding LuxR-type (LAL) regulatory gene, metabolic remodeling, and cell-to-cell interaction. These new approaches may enable the identification of novel and/or poorly understood antibiotics, such as piperidamycin and stambomycin, and are all characterized by applicability to a wide range of actinomycetes and potential scalability to high throughput.

Streptomyces种中的激活潜在生物合成基因簇的有用方法其中就包括核糖体工程:在培养基内添加N-乙酰葡糖胺或者进行dasR基因敲除(这种基因负责编码N-乙酰葡糖胺应答调节蛋白)大型的ATP结合性LuxR型调节基因特异性途径的结构性过表达,代谢改造以及胞间相互作用。这些新兴方法能够发现一些新型和/或者了解不全面的抗生素,就如piperidamycin和stambomycin,能够适用于大部分放线菌以及具有高流通量可测性的特征。

分类: 英语

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