Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation.
Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation.
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DOI：
10.1186/s12934-022-01958-3
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发表时间：
2022-11-03
影响因子：
6.4
通讯作者：
Ward, John M.
中科院分区：
文献类型：
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作者：
Allen, James R.;Torres-Acosta, Mario A.;Mohan, Naresh;Lye, Gary J.;Ward, John M.
通过生物路线生产化学品受到简单易用的稳定系统的限制。在文献中可以找到广泛的质粒稳定机制，然而，这些机制如何影响遗传稳定性以及宿主菌株如何恢复到非生产性变体在单细胞水平上知之甚少。这种现象可能会在生产规模的生物反应器中造成困难，因为可能会出现不同群体的生产和非生产细胞。为了了解如何防止非生产性菌株的出现，在单细胞水平上了解菌株行为是至关重要的。位于质粒载体上的基因的持久性取决于许多因素，但大致可分为结构稳定性和分离稳定性。结构稳定性指的是细胞抵抗基因突变导致生产途径中基因功能丧失的能力，分离稳定性指的是细胞正确地将质粒分布到子细胞中以保持拷贝数的能力。缺乏分离稳定性会在复制过程中迅速产生无质粒变异，从而影响生产力。柠檬酸合成酶在操纵子中的表达与荧光报告基因的表达相联系，从而能够快速筛选大肠杆菌连续发酵中模型化学合成途径的保留。没有额外的质粒稳定化的细胞在进入连续阶段后立即开始失去生产力。包括多聚体解析站点CER，在检测到生产率下降之前，实现了58小时的稳定生产周期。单细胞荧光测量表明，在没有CER稳定的情况下，无质粒变异体迅速出现，这可能是由于在细胞分裂过程中，质粒不均匀地分布到子细胞中所致。添加CER可使总化学产率提高50%以上。这项研究表明，如果质粒得到正确的稳定，它们在工业生物化学品生产中用作途径载体的潜力仍然很大。我们证明了需要可获得的细菌“工具箱”，以便能够快速生产已知的、稳定的细菌生产菌株，从而能够为化学工业提供连续的规模化发酵。网上版载有补充材料，可在10.1186/s12934-022-01958-3查阅。
The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial ‘toolkits’ to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry. The online version contains supplementary material available at 10.1186/s12934-022-01958-3.
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