Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection
Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection
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DOI：
10.1128/spectrum.00259-21
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发表时间：
2021-07
影响因子：
3.7
通讯作者：
Masahito Ishikawa;Takaaki Kojima;K. Hori
中科院分区：
文献类型：
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作者：
Masahito Ishikawa;Takaaki Kojima;K. Hori
宝石对包括环境保护在内的各种应用都是有益的。然而，创业板释放到环境中的风险已经争论了很长时间。摘要生物遏制是防止基因工程微生物在环境中不受控制地扩散的一种保障策略。含有生物的宝石被设计成只能在特定分子存在的情况下存活。设计一种污染物降解和污染物依赖的宝石，防止其在清洁环境后扩散。在本研究中，我们提出了一种基于不动杆菌属的甲苯生物降解菌。Tol 5的染色体上缺失了编码必需外膜蛋白的BAMA基因，但与携带受Pu启动子和调节蛋白XylR调控的BAMA基因的质粒互补。该菌株对甲苯的降解与野生型TOL5相似。尽管甲苯耗尽后，pUBamA菌株的细胞生长受到显著抑制，但逃逸突变的出现频率为1/5 3×10−7细胞。逃逸突变体的分析表明，携带启动子的插入序列(ISS)被插入到Pu启动子和互补质粒上的BAMA基因之间。对从逃逸突变体中提取的质粒进行Minion深度测序后，能够确定与逃逸突变体的出现有关的三种ISS，从而提出了减少逃逸突变体的策略。重要的宝石对包括环境保护在内的各种应用都有好处。然而，创业板释放到环境中的风险已经争论了很长时间。如果一种污染物被用作生物遏制系统的特定分子，那么能够降解污染物的宝石可以用于环境保护。然而，据我们所知，到目前为止，学术期刊上还没有关于真正污染物的生物降解剂的报道。这可能是因为降解污染物的酶在易驯化的细菌(如大肠杆菌)中难以表达。另一方面，由于缺乏基因操作工具，具有降解污染物的酶的细菌作为一大批宝石往往很难处理。这项研究报告了甲苯降解器生物遏制策略的可行性。我们的研究结果为构建环境保护的宝石生物遏制系统提供了有益的启示。
GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. ABSTRACT Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10−7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.