Prokaryotes modified stably on the genome are of great importance for production of fine and commodity chemicals. Traditional methods for genome engineering have long suffered from imprecision and low efficiencies, making construction of suitable high-producer strains laborious. Here, we review the recent advances in discovery and refinement of molecular precision engineering tools for genome-based metabolic engineering in bacteria for chemical production, with focus on the λ-Red recombineering and the clustered regularly interspaced short palindromic repeats/Cas9 nuclease systems. In conjunction, they enable the integration of in vitro-synthesized DNA segments into specified locations on the chromosome and allow for enrichment of rare mutants by elimination of unmodified wild-type cells. Combination with concurrently developing improvements in important accessory technologies such as DNA synthesis, high-throughput screening methods, regulatory element design, and metabolic pathway optimization tools has resulted in novel efficient microbial producer strains and given access to new metabolic products. These new tools have made and will likely continue to make a big impact on the bioengineering strategies that transform the chemical industry.

在基因组上稳定修饰的原核生物对于生产精细化学品和商品化学品至关重要。长期以来，用于基因组工程的传统方法一直受制于不精确且效率低下的问题，这使得构建合适的高产菌株变得费力。在这里，我们回顾了在细菌生产中基于基因组的代谢工程的分子精密工程工具的发现和完善方面的最新进展，重点是λ-Red重组和聚集的规则间隔的短回文重复序列/ Cas9核酸酶系统。结合起来，它们可以将体外合成的DNA片段整合到染色体上的指定位置，并通过消除未修饰的野生型细胞来富集稀有突变体。代谢途径优化工具已产生了新型有效的微生物生产菌株，并获得了新的代谢产物。这些新工具已经并且可能会继续对改变化学工业的生物工程策略产生巨大影响。