Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2012 Aug 21;109(34):13538-43. doi: 10.1073/pnas.1206346109. Epub 2012 Aug 6.
Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.
尽管微生物代谢工程传统上依赖于理性和知识驱动的技术，但通过利用表型多样化和筛选的组合方法，可以进一步提高菌株的性能。在这里，我们展示了全局转录机制工程和高通量 L-酪氨酸筛选的联合使用，以提高大肠杆菌中 L-酪氨酸的产量。该方法成功地从两个独立的诱变文库（rpoA 和 rpoD）中生成了三个菌株，与已经具有高产能力的合理工程化亲本菌株相比，L-酪氨酸滴度提高了 114%。通过转录分析和全基因组测序进行的后续菌株表征允许从明确的突变完全重建表型，并指出大肠杆菌的酸应激抗性途径和严格响应在赋予该表型方面的重要作用。因此，这项研究提供了细胞全测量有助于阐明工程化细胞特性的遗传和生化基础的首批实例之一，导致特定染色体突变导致代谢物过量产生的完全恢复。
