The ability of DNA to produce a functional protein even after transfer to a foreign host is of fundamental importance in both evolutionary biology and biotechnology, enabling horizontal gene transfer in the wild and heterologous expression in the lab. However, the influence of genetic particulars on DNA functionality in a new host is poorly understood, as are the evolutionary mechanisms of assimilation and refinement. Here, we describe an automation-enabled large-scale experiment wherein Escherichia coli strains were evolved in parallel after replacement of the genes pgi or tpiA with orthologous DNA from donor species spanning all domains of life, from humans to hyperthermophilic archaea. Via analysis of hundreds of clones evolved for 50,000+ cumulative generations across dozens of independent lineages, we show that orthogene-upregulating mutations can completely mitigate fitness defects that result from initial non-functionality, with coding sequence changes unnecessary. Gene target, donor species and genomic location of the swap all influenced outcomes—both the nature of adaptive mutations (often synonymous) and the frequency with which strains successfully evolved to assimilate the foreign DNA. Additionally, time series DNA sequencing and replay evolution experiments revealed transient copy number expansions, the contingency of lineage outcome on first-step mutations and the ability for strains to escape from suboptimal local fitness maxima. Overall, this study establishes the influence of various DNA and protein features on cross-species genetic interchangeability and evolutionary outcomes, with implications for both horizontal gene transfer and rational strain design.

即使在转移到外源宿主后，DNA产生功能蛋白的能力在进化生物学和生物技术中也至关重要，这使得在实验室中野生和异源表达水平基因转移成为可能。但是，人们对遗传特性对新宿主中DNA功能的影响以及同化和精炼的进化机制了解甚少。在这里，我们描述了一个自动化的大规模实验，其中在替换基因pgi或tpiA后并行进化了大肠杆菌菌株从人类到超嗜热古细菌的供体物种的直系同源DNA。通过对数十个独立谱系中进化为50,000+累积世代的数百个克隆的分析，我们表明，正基因上调突变可以完全缓解由初始无功能性导致的适应性缺陷，而无需更改编码序列。交换的基因靶标，供体种类和基因组位置均影响结果-适应性突变的性质（通常是同义词）以及菌株成功进化以吸收外源DNA的频率。此外，时间序列DNA测序和重播进化实验显示瞬时拷贝数扩展，第一步突变的谱系结果的偶然性，以及菌株从次优局部适应度最大值中逃脱的能力。总体而言，这项研究建立了各种DNA和蛋白质特征对跨物种遗传互换性和进化结果的影响，同时对水平基因转移和合理的菌株设计都具有影响。