Carbon-loss-guided metabolic engineering improved carbon atom economy and industrial feedstock utilization for l-threonine fermentation.

l-Threonine is a high-value amino acid widely used in the feed, food, and pharmaceutical industries, but its industrial production is limited by low yield. In this study, whole-genome sequencing of industrial strain Thr1 was performed, and the genome-scale metabolic model GSMM-iJHL1061 was reconstructed. The model identified 54 CO2-related reactions and predicted 0.89 mol CO2 release per mol l-threonine synthesized, mainly from the PPP and TCA cycle. Attenuation of decarboxylation reactions involving ilvBN, lysA, gnd, and icd, combined with RuBisCO-Prk and cynT-pyc-based CO2 fixation, increased the yield to 0.66 g/g. Deletion of 166 non-essential genes generated Thr9, increasing the yield to 0.67 g/g. Further introduction of maltose hydrolysis and sucrose utilization systems generated Thr10, which produced 132.5 g/L l-threonine in a 1000-L fermenter, with a yield of 0.68 g/g. Overall, l-threonine yield was substantially improved through systems metabolic engineering, providing a useful reference for constructing high-yield microbial cell factories.