Multi-Functional Enzymes and Metabolic Cross-Feeding Constrain Implementation of Genome Scale Metabolic Model Based Designs

Genome scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling, pairing product formation with substrate utilization. While such approaches can improve strain performance beyond levels typically accessible using targeted strain engineering approaches, sustainable feedstocks often pose a challenge for GSMM-based methods due to incomplete underlying metabolic data. Specifically, we address a four-gene deletion design for the lignin-derived non-sugar carbon source, para-coumarate, that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product. Through omics, promoter-variation and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism is rate-limited by a specific fumarate hydratase isomer (PP_0897) in the citrate cycle. A metabolic cross-feeding experiment with the completed design strain reveals broader functions for this enzyme beyond its presumed annotation. Finally, a double sensitivity analysis demonstrates a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. This work highlights the challenge of precisely inactivating metabolic reactions encoded in multifunctional proteins.

全基因组规模代谢模型（Genome-scale Metabolic Models，GSMM）常被用于筛选可引发生长偶联的基因敲除组合，即将产物生成与底物利用相耦联。尽管此类策略可将菌株性能提升至定向菌株工程手段通常难以企及的水平，但基于GSMM的方法往往受限于可持续原料相关的底层代谢数据不完备问题。

具体而言，本研究针对木质素来源的非糖碳源对香豆酸（para-coumarate）设计了四基因敲除方案，但该方案此前被证实难以实施。本研究对完整实施的对香豆酸转化为谷氨酰胺的方案性能进行了表征，谷氨酰胺是一类极具应用价值的生物制造中间体。本研究中，谷氨酰胺可进一步转化为靛素（indigoidine）——一种兼具可持续性的替代色素与模式异源产物。

通过对完整实施的基因敲除设计开展组学分析、启动子变异验证与生长特性表征，本研究证实：在三羧酸循环中，特定延胡索酸水合酶（fumarate hydratase）同工型（PP_0897）是芳香族物质分解代谢的限速步骤。针对该工程菌株开展的代谢交叉供养实验显示，该酶的功能远超其初始注释所推测的范围。最后，双敏感性分析结果表明，在生长偶联设计中所有基因均已敲除的工程菌株内，延胡索酸水合酶活性是不可或缺的。本研究凸显了精准失活多功能蛋白质所编码的代谢反应所面临的挑战。