Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Nucleic Acids Res. 2022 Jun 10;50(10):e58. doi: 10.1093/nar/gkac094.
Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5-10 kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli's utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.
定向进化是一种在缺乏详细的序列-功能关系的情况下对生物学进行工程改造的强大方法。为了能够对由多基因途径编码的复杂表型进行定向进化，我们需要具有 >5-10kb 长度的 DNA 序列的大型文库，消除基因组搭便车突变，并将多样化和筛选步骤解耦。为了应对这些挑战，我们开发了诱导定向进化 (IDE)，它使用温和噬菌体来包装大质粒，并在细胞内诱变后将其转移到未成熟的细胞中。为了证明 IDE，我们从地衣芽孢杆菌进化了一个 5 基因途径，该途径加速了大肠杆菌中塔格糖的分解代谢，导致在仅两轮进化后，在塔格糖上生长时，克隆的滞后时间缩短了 65%。接下来，我们从短双歧杆菌 UC2003 进化了一个 15.4kb、10 基因途径，帮助大肠杆菌利用密二糖。经过三轮 IDE，我们分离出了进化途径，它们不仅将滞后时间缩短了两倍以上，而且使最终光密度提高了 150%。总之，这项工作提高了大肠杆菌中整个途径定向进化方法的能力和实用性。
