Department of Chemical & Biological Engineering, Tufts University, Medford, MA, 02155, USA.
Kcat Enzymatic Private Limited, Bengaluru, Karnataka, 560005, India.
Metab Eng. 2023 Sep;79:14-26. doi: 10.1016/j.ymben.2023.07.001. Epub 2023 Jul 4.
Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3p), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3p (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3p occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3p to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3p-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.
在经过验证的工业微生物（如酿酒酵母）中，工程利用非天然底物或合成异养作用，代表了一种将丰富可再生的碳和能源来源转化为生物过程输入的机会。我们之前证明，在非天然底物木糖上生长时，激活该酵母在利用半乳糖生长时用于协调底物利用和生物量形成的半乳糖（GAL）调控系统，会导致与同异性代谢途径的组成型过表达相比，基因表达谱发生巨大改变并生长更快。然而，这项工作涉及到创建一个木糖诱导型 Gal3p（Gal3p）变体，Gal3p 是 GAL 调控系统的传感器蛋白，这使得这种半合成调控系统难以适应其他非天然底物。在这里，我们报告了一种变体 Gal3p（代谢协调器）的构建，该变体在存在结构多样的底物时表现出强大的 GAL 调控系统激活作用，并再现了天然系统的动力学。多项分子建模研究表明，Gal3p 以非诱导依赖的方式占据与结合半乳糖的 Gal3p 对应的构象状态。使用 Gal3p 测试非天然木质纤维素糖木糖、阿拉伯糖和纤维二糖的同化的调控系统方法与相同一组代谢基因的组成型过表达相比，可获得更高的生长速率和最终细胞密度。随后，对所有三种非天然底物的快速和完全共利用的证明表明，Gal3p 介导的 GAL 调控系统激活的动态全局基因表达变化可能对工程合成异养作用普遍有益。
