Construction of a GTP regeneration system by regulating gene expression in the pathway

Abstract

Guanosine 5′-triphosphate (GTP) provides precursors for the biological synthesis of various valuable compounds. However, the insufficient supply of GTP is often encountered when engineering microbes for the synthesis of certain products, which results in metabolic imbalance and unsatisfying product yield. To tackle this issue, a general strategy for improving GTP regeneration was developed in this study. The effects of dosage and combination strategy of genes related to GTP synthesis were examined to pinpoint the most critical genes influencing GTP production in Escherichia coli. A regeneration system of GTP was derived by the overexpression of genes guaA and guaB, the intracellular GTP concentration of which improved by 81%. To further verify the applicability of the engineered GTP regeneration system on strengthening the product synthesis, it was applied to the production of riboflavin and colanic acid that required GTP as a precursor. It turned out that the titer of riboflavin was increased by 95.8% and the titer of colanic acid was increased by 44%. The GTP regeneration system developed here can also be applied to other products that use GTP as a precursor or require GTP as a cofactor.