Engineering the intracellular lifetime of pathway enzymes to enhance microbial myo-inositol production.

The continuous turnover of pathway enzymes imposes a significant metabolic burden on engineered cells and often limits production efficiency. However, enzyme lifetime is rarely considered a fundamental parameter in pathway design and optimization. Here, using the myo-inositol biosynthetic pathway in Escherichia coli as a model, which consists of myo-inositol 1-phosphate synthase (IPS) and inositol monophosphatase (IMP), we demonstrate that selective regulation of the intracellular half-lives of pathway enzymes can enhance myo-inositol production. Specifically, fusing a degradation-protective short peptide, termed long-tail 1 (lt1) to the C-terminus of the rate-limiting enzyme IPS prolonged its intracellular half-life from 24 min to 135 min without compromising enzymatic activity, leading to a 30% increase in myo-inositol titer. In contrast, stabilizing IMP via the same tag reduced overall production due to intensified substrate competition for glucose-6-phosphate. These results highlight the importance of tailoring enzyme lifetime according to pathway topology. Mechanistically, lt1 fusion protects the tagged enzyme from ClpXP-mediated proteolysis by masking C-terminal degrons. Combining lt1 tagging with ClpXP deletion further improved the myo-inositol titer by 48%. Our findings establish rational control of enzyme lifetime as a general and complementary strategy to conventional expression-based approaches for optimizing microbial cell factories.