Renda Brian A, Dasgupta Aurko, Leon Dacia, Barrick Jeffrey E
Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, USA.
Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, USA
J Bacteriol. 2015 Mar;197(5):872-81. doi: 10.1128/JB.02263-14. Epub 2014 Dec 15.
Acinetobacter baylyi ADP1 has the potential to be a versatile bacterial host for synthetic biology because it is naturally transformable. To examine the genetic reliability of this desirable trait and to understand the potential stability of other engineered capabilities, we propagated ADP1 for 1,000 generations of growth in rich nutrient broth and analyzed the genetic changes that evolved by whole-genome sequencing. Substantially reduced transformability and increased cellular aggregation evolved during the experiment. New insertions of IS1236 transposable elements and IS1236-mediated deletions led to these phenotypes in most cases and were common overall among the selected mutations. We also observed a 49-kb deletion of a prophage region that removed an integration site, which has been used for genome engineering, from every evolved genome. The comparatively low rates of these three classes of mutations in lineages that were propagated with reduced selection for 7,500 generations indicate that they increase ADP1 fitness under common laboratory growth conditions. Our results suggest that eliminating transposable elements and other genetic failure modes that affect key organismal traits is essential for improving the reliability of metabolic engineering and genome editing in undomesticated microbial hosts, such as Acinetobacter baylyi ADP1.
拜氏不动杆菌ADP1具有成为合成生物学通用细菌宿主的潜力，因为它具有天然可转化性。为了检验这一理想特性的遗传可靠性，并了解其他工程能力的潜在稳定性，我们在富含营养的肉汤中使ADP1传代培养1000代，并通过全基因组测序分析所发生的遗传变化。在实验过程中，可转化性大幅降低，细胞聚集性增加。在大多数情况下，IS1236转座元件的新插入和IS1236介导的缺失导致了这些表型，并且在所选突变中总体上较为常见。我们还观察到一个前噬菌体区域发生了49 kb的缺失，该缺失从每个进化的基因组中移除了一个已用于基因组工程的整合位点。在经过7500代减少选择传代培养的谱系中，这三类突变的发生率相对较低，表明它们在常见实验室生长条件下提高了ADP1的适应性。我们的结果表明，消除影响关键生物体性状的转座元件和其他遗传故障模式对于提高未驯化微生物宿主（如拜氏不动杆菌ADP1）中代谢工程和基因组编辑的可靠性至关重要。
