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

该项目的目标是重新设计生物生产过程中酶的变构调节，以实现自我调节和动态代谢控制。使用高丝氨酸脱氢酶（HSD）和赖氨酸生物合成作为一个模型系统，我们将实施赖氨酸作为一个信号分子来控制代谢通量的苏氨酸途径，这是必要的细胞生长，但不希望赖氨酸生产。将设计具有修饰的变构调节的不同HSD变体。通过构建携带突变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后的通量再分布。因此，HSD的酶活性应根据生物过程中细胞的生理条件，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，从而允许足够的通量流向苏氨酸途径以用于细胞生长。随着生产阶段赖氨酸浓度的增加，HSD的抑制作用将自动增强，从而底物将被引导到赖氨酸生产途径中。通过这种新方法，我们将开发一种新的工具，用于克服与传统菌株开发中的基因过表达或敲除等剧烈遗传修饰相关的一些主要问题。

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.