Table_2_Mutations identified in engineered Escherichia coli with a reduced genome.XLSX

Characterizing genes that regulate cell growth and survival in model organisms is important for understanding higher organisms. Construction of strains harboring large deletions in the genome can provide insights into the genetic basis of cell growth compared with only studying wild-type strains. We have constructed a series of genome-reduced strains with deletions spanning approximately 38.9% of the E. coli chromosome. Strains were constructed by combining large deletions in chromosomal regions encoding nonessential gene groups. We also isolated strains Δ33b and Δ37c, whose growth was partially restored by adaptive laboratory evolution (ALE). Genome sequencing of nine strains, including those selected following ALE, identified the presence of several Single Nucleotide Variants (SNVs), insertions, deletions, and inversions. In addition to multiple SNVs, two insertions were identified in ALE strain Δ33b. The first was an insertion at the promoter region of pntA, which increased cognate gene expression. The second was an insertion sequence (IS) present in sibE, encoding the antitoxin in a toxin-antitoxin system, which decreased expression of sibE. 5 strains of Δ37c independently isolated following ALE harboring multiple SNVs and genetic rearrangements. Interestingly, a SNV was identified in the promoter region of hcaT in all five strains, which increased hcaT expression and, we predict, rescued the attenuated Δ37b growth. Experiments using defined deletion mutants suggested that hcaT encodes a 3-phenylpropionate transporter protein and is involved in survival during stationary phase under oxidative stress. This study is the first to document accumulation of mutations during construction of genome-reduced strains. Furthermore, isolation and analysis of strains derived from ALE in which the growth defect mediated by large chromosomal deletions was rescued identified novel genes involved in cell survival.

在模式生物（model organisms）中鉴定调控细胞生长与存活的基因，对于理解高等生物的生命机制具有重要意义。相较于仅研究野生型菌株（wild-type strains），构建基因组携带有大片段缺失的菌株，可为解析细胞生长的遗传基础提供全新视角。我们构建了一系列基因组精简菌株（genome-reduced strains），其缺失片段覆盖大肠杆菌（E. coli）染色体约38.9%的区域。上述菌株通过整合染色体区域内编码非必需基因群的大片段缺失构建得到。

我们还分离得到了Δ33b与Δ37c菌株，二者的生长缺陷可通过适应性实验室进化（adaptive laboratory evolution, ALE）得到部分恢复。对包括经过适应性实验室进化筛选得到的菌株在内的9株菌株进行基因组测序，共鉴定到多个单核苷酸变异（Single Nucleotide Variants, SNVs）、插入突变、缺失突变与染色体倒位事件。除多组单核苷酸变异外，在经过适应性实验室进化的Δ33b菌株中还鉴定到两处插入突变：第一处插入突变位于pntA的启动子区域，可上调该基因的转录表达水平；第二处为位于sibE基因内的插入序列（insertion sequence, IS），sibE编码毒素-抗毒素系统（toxin-antitoxin system）中的抗毒素蛋白，该插入序列可下调sibE的基因表达。

经适应性实验室进化独立分离得到的5株Δ37c菌株，均携带有多组单核苷酸变异与遗传重排事件。值得注意的是，在全部5株Δ37c菌株的hcaT基因启动子区域均鉴定到一处单核苷酸变异，该变异可提升hcaT的表达水平，我们据此推测其可恢复减毒的Δ37b菌株的生长缺陷。通过定点缺失突变株（defined deletion mutants）开展的实验表明，hcaT编码一种3-苯基丙酸（3-phenylpropionate）转运蛋白，并参与氧化应激（oxidative stress）条件下稳定期（stationary phase）的细胞存活过程。

本研究首次报道了基因组精简菌株构建过程中突变的积累现象。此外，我们通过适应性实验室进化获得了可恢复大片段染色体缺失所致生长缺陷的菌株，并对其进行分离与分析，由此鉴定出一批参与细胞存活过程的新型基因。