Engineering Corynebacterium glutamicum for high-titer biosynthesis of hyaluronic acid

Hyaluronic acid (HA) is a member of the glycosaminoglycan family and has been widely used in the clinical, medical, cosmetic and food industries. In this study, we constructed a superior cell factory in Corynebacterium glutamicum for high-titer HA biosynthesis through systematic design and metabolic engineering based on a genome-scale metabolic model, iCW773. The OptForce_MUST algorithm was used in iCW773 to determine genetic interventions by using flux balance analysis. Enhancement of the HA biosynthesis pathway and attenuation of the glycolysis pathway, the pentose phosphate pathway and the dehydrogenation of pyruvate were predicted as targets for genetic modulations. Various genetic strategies were employed, including an additional promoter, P_dapB, driving hasB expression, antisense RNA-mediated attenuation of fba, zwf deletion and lactate/acetate pathway knockout. The integrated genetic changes in recombinant C. glutamicum produced 24.5 g/L HA in a fed-batch culture. Finally, pyruvate dehydrogenase activity was further reduced by antisense RNA and initial codon mutation to divert carbon flux from byproducts to HA. The corresponding modified strain, CgHA25, achieved a titer of 28.7 g/L. The byproduct concentration was reduced by half, and the major weight-average molecular weight (M_w) component was 0.21 MDa. This work reports a significant improvement in the HA titer in a safe host achieved by systematic metabolic engineering.