Redesign of allosteric regulation of enzyme for self-regulated dynamic control of metabolic fluxes in microbial amino acid production

本项目的目标是重新设计酶的变构调节(S)，以实现生物生产过程中的自我调节和动态代谢控制。利用高丝氨酸脱氢酶(HSD)和赖氨酸生物合成作为模型系统，我们将实现赖氨酸作为信号分子来控制苏氨酸途径的代谢流量，苏氨酸途径是细胞生长所必需的，但不是赖氨酸生产所需要的。将设计不同的HSD变体，并修改变构调节。通过构建携带突变的HSD的谷氨酸棒杆菌突变体，我们将使用代谢和通量分析来研究遗传扰动下的通量重分配。在我们的方法中，HSD的酶活性应该根据生物过程中的细胞生理条件，特别是赖氨酸浓度进行调节。在细胞生长阶段，细胞内赖氨酸浓度较低，因此HSD的抑制作用不会很强，从而允许足够的流量进入细胞生长的苏氨酸途径。随着生产阶段赖氨酸浓度的增加，HSD的抑制作用会自动增强，从而引导底物进入赖氨酸产生的途径。通过这种新的方法，我们将开发一种新的工具来克服与剧烈的遗传修改相关的一些主要问题，如传统菌株开发中的基因过度表达或基因敲除。

The goal of this project is to redesign allosteric regulation of enzyme(s) for self-regulated and dynamic metabolic control in bioproduction processes. Using homoserine dehydrogenase (HSD) and lysine biosynthesis as a model system, we will implement lysine as a signalling molecule to control metabolic flux to the threonine pathway which is necessary for cell growth but undesired for lysine production. Different HSD variants with modified allosteric regulation will be designed. By construction of Corynebacterium glutamicum mutants bearing the mutated HSD, we will use metabolic and flux analyses to investigate the flux redistribution upon the genetic perturbation. With our approach, the enzyme activity of HSD should be adjusted according to the cellular physiological conditions during the bioprocess, especially to lysine concentration. At the stage of cell growth, the intracellular lysine concentration is low, and thus the inhibition of HSD will not be strong, allowing enough flux to the threonine pathways for cell growth. With the increase of lysine concentration in the production phase, the inhibition of HSD will be automatically enhanced and thus the substrate will be channelled into the pathway of lysine production. With this novel approach, we will develop a new tool for overcoming some of the major problems associated with drastic genetic modifications like gene overexpression or knockout in conventional strain development.