Lanza Amanda M, Alper Hal S
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
Methods Mol Biol. 2011;765:253-74. doi: 10.1007/978-1-61779-197-0_15.
Cellular hosts are widely used for the production of chemical compounds including pharmaceutics, fuels, and specialty chemicals. Strain engineering focuses on manipulating and improving these hosts for new and enhanced functionalities including increased titers and better bioreactor performance. These tasks have traditionally been accomplished using a combination of random mutation, screening and selection, and metabolic engineering. However, common metabolic engineering techniques are limited in their capacity to elicit multigenic, complex phenotypes. These phenotypes can also include nonpathway-based traits such as tolerance and productivity. Global transcription machinery engineering (gTME) is a generic methodology for engineering strains with these complex cellular phenotypes. In gTME, dominant mutant alleles of a transcription-related protein are screened for their ability to reprogram cellular metabolism and regulation, resulting in a unique and desired phenotype. gTME has been successfully applied to both prokaryotic and eukaryotic systems, resulting in improved environmental tolerances, metabolite production, and substrate utilization. The underlying principle involves creating mutant libraries of transcription factors, screening for a desired phenotype, and iterating the process in a directed evolution fashion. The successes of this approach and details for its implementation and application are described here.
细胞宿主被广泛用于生产包括药物、燃料和特殊化学品在内的化合物。菌株工程专注于操纵和改进这些宿主，以实现新的和增强的功能，包括提高滴度和改善生物反应器性能。传统上，这些任务是通过随机突变、筛选和选择以及代谢工程的组合来完成的。然而，常见的代谢工程技术在引发多基因、复杂表型方面的能力有限。这些表型还可能包括基于非途径的特性，如耐受性和生产力。全局转录机制工程（gTME）是一种用于构建具有这些复杂细胞表型菌株的通用方法。在gTME中，筛选转录相关蛋白的显性突变等位基因，以评估其重新编程细胞代谢和调控的能力，从而产生独特且理想的表型。gTME已成功应用于原核生物和真核生物系统，提高了环境耐受性、代谢物产量和底物利用率。其基本原理包括创建转录因子突变文库、筛选所需表型，并以定向进化的方式重复该过程。本文描述了这种方法的成功之处及其实施和应用的细节。
