Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824.
Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824.
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DOI：
10.1186/s13068-015-0410-0
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发表时间：
2016
影响因子：
6.3
通讯作者：
Minton NP
中科院分区：
文献类型：
--
作者：
Ehsaan M;Kuit W;Zhang Y;Cartman ST;Heap JT;Winzer K;Minton NP
丙酮丁醇梭菌代表了生物燃料生物丁醇工业生产的范例基础，也是代谢工程的焦点。我们之前已经开发了基于使用pyrE和codA基因作为反选择标记在病原体艰难梭菌中创建框内、无标记缺失突变体的程序。在当前的研究中，我们试图测试它们在丙酮丁醇梭菌中的适用性。两个系统都可以轻松分离丙酮丁醇梭菌 ATCC 824 spo0A 和 cac824I 基因的框内删除，分别导致孢子形成负表型和改进的转化。基于pyrE的系统还用于灭活假定的糖原合酶（CA_C2239，glgA）和pSOL1淀粉酶基因（CA_P0168，amyP），分别导致颗粒糖和淀粉酶的产生不足。它们的分离提供了利用pyrE系统的关键优势之一的机会，即能够以基因组中适当的基因剂量快速补充突变的能力。在这两种情况下，它们的表型在颗粒糖 (glgA) 和淀粉酶 (amyP) 的产生方面都得到了恢复。使用的 ATCC 824 COSMIC 联盟实验室菌株的基因组重测序显示存在 177 个 SNV 和 49 个 Indel，其中包括 pSOL1 巨质粒中的 4916 bp 缺失。随后显示，从 ATCC 重新获得（2011 年 11 月）的 824 个菌株中存在总共 175 个 SNV 和 48 个插入缺失，因此，很可能是已发表的基因组序列 NC_003030（染色体）和 NC_001988 (pSOL1) 中的错误。 codA或pyrE反选择标记在分离缺失突变体方面似乎同样有效，但使用pyrE突变体作为宿主具有相当大的优点，因为通过使用ACE（等位基因偶联交换）载体，创建的突变体（通过任何方式）可以在pyrE等位基因的恢复的同时快速补充。这避免了高拷贝数质粒经常观察到的表型效应，并且无需添加抗生素以确保质粒保留。我们的研究还揭示了 ATCC 824 基因组序列中惊人数量的错误，同时强调需要对常用的实验室菌株进行重新测序。本文的在线版本 (doi:10.1186/s13068-015-0410-0) 包含补充材料，可供授权用户使用。
Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum. Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1). The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Our study also revealed a surprising number of errors in the ATCC 824 genome sequence, while at the same time emphasising the need to re-sequence commonly used laboratory strains. The online version of this article (doi:10.1186/s13068-015-0410-0) contains supplementary material, which is available to authorized users.