Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold