Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.
Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.
复制标题
基因组减少的枯草芽孢杆菌菌株的表征及其在鸟苷和胸苷生产中的应用
DOI：
10.1186/s12934-016-0494-7
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发表时间：
2016-06-03
影响因子：
6.4
通讯作者：
Zhao X
中科院分区：
文献类型：
--
作者：
Li Y;Zhu X;Zhang X;Fu J;Wang Z;Chen T;Zhao X
基因组精简已成为提高生物基产品生产效率的一种有效策略。人们已经做出了许多努力，通过减小微生物的基因组大小来构建理想的底盘细胞。据报道，基因组减小的枯草芽孢杆菌MBG874菌株在生产包括碱性纤维素酶和蛋白酶在内的几种异源酶方面显示出明显优势。除了酶之外，枯草芽孢杆菌也用于化学品的生产。据我们所知，基因组精简是否可用于优化核苷类产品等化学品的生产仍然未知。
在这项研究中，我们通过删除枯草芽孢杆菌168染色体上的非必需区域构建了一系列基因组精简菌株。这些基因组精简大小在581.9到814.4kb之间的菌株显示出生长速率、芽孢形成率、转化效率和维持系数显著降低，以及细胞产量增加。我们对基因组精简菌株进行了重新改造，使其分别生产鸟苷和胸苷。在purA被敲除且prs、purF和guaB共过量表达的菌株BSK814G2中，产生了115.2mg/L的鸟苷，与将相同基因改造引入亲本菌株构建的对照菌株相比，产量提高了4.4倍。我们还通过在菌株BSK756中删除tdk基因并过量表达来自大肠杆菌的prs、ushA、thyA、dut和ndk基因构建了一个胸苷生产菌株，所得菌株BSK756T3积累了151.2mg/L的胸苷，与相应对照菌株相比增加了5.2倍。
基因组规模的遗传操作对枯草芽孢杆菌的生理特性和细胞代谢有多种影响。通过分别引入与鸟苷和胸苷积累相关的特定基因改造，我们证明了与野生型菌株相比，基因组精简菌株作为生产这两种产品的底盘细胞具有极大改善的性能。这些菌株在生产其他核苷和类似衍生化学品方面也具有很大潜力。
本文的在线版本（doi:10.1186/s12934 - 016 - 0494 - 7）包含补充材料，授权用户可获取。
Background:Genome streamlining has emerged as an effective strategy to boost the production efficiency of bio-based products. Many efforts have been made to construct desirable chassis cells by reducing the genome size of microbes. It has been reported that the genome-reduced Bacillus subtilis strain MBG874 showed clear advantages for the production of several heterologous enzymes including alkaline cellulase and protease. In addition to enzymes, B. subtilis is also used for the production of chemicals. To our best knowledge, it is still unknown whether genome reduction could be used to optimize the production of chemicals such as nucleoside products.Results:In this study, we constructed a series of genome-reduced strains by deleting non-essential regions in the chromosome of B. subtilis 168. These strains with genome reductions ranging in size from 581.9 to 814.4 kb displayed markedly decreased growth rates, sporulation ratios, transformation efficiencies and maintenance coefficients, as well as increased cell yields. We re-engineered the genome-reduced strains to produce guanosine and thymidine, respectively. The strain BSK814G2, in which purA was knocked out, and prs, purF and guaB were co-overexpressed, produced 115.2 mg/L of guanosine, which was 4.4-fold higher compared to the control strain constructed by introducing the same gene modifications into the parental strain. We also constructed a thymidine producer by deleting the tdk gene and overexpressing the prs, ushA, thyA, dut, and ndk genes from Escherichia coli in strain BSK756, and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine, showing a 5.2-fold increase compared to the corresponding control strain.Conclusions:Genome-scale genetic manipulation has a variety of effects on the physiological characteristics and cell metabolism of B. subtilis. By introducing specific gene modifications related to guanosine and thymidine accumulation, respectively, we demonstrated that genome-reduced strains had greatly improved properties compared to the wild-type strain as chassis cells for the production of these two products. These strains also have great potential for the production of other nucleosides and similar derived chemicals.