Efficient and Economic Utilization of Cellobiose for Glycosylation Modification by Regulating Carbon Source Supply and Metabolic Pathway In Vivo

Abstract

Glycosylation, one of the most common and significant modifications in nature, has prompted the development of a cellobiose phosphorolysis route for glycosylation in vivo. However, the process of glycosylation is hampered by the notably low conversion rate of cellobiose. In this work, regulation of the carbon source supply by changing the ratio of glucose to cellobiose improved the conversion rate of cellobiose, resulting in enhancing the efficiency of glycosylation and the production of vitexin. Moreover, three genes (pgm, agp, and ushA) involved in the degradation of UDP-glucose were knocked out to relieve the degradation and diversion of the cellobiose phosphorolysis route. Finally, through the optimization of conversion conditions, we observed a continuous enhancement in cellobiose conversion rate and vitexin production in BL21ΔushAΔagp-TcCGT-CepA, corresponding to an increased concentration of added glucose. The maximum production of vitexin reached 2228 mg/L with the addition of 2 g/L cellobiose and 6 g/L glucose, which was 312% of that in BL21-TcCGT-CepA with the addition of 2 g/L cellobiose. The conversion rate of cellobiose in BL21ΔushAΔagp-TcCGT-CepA reached 88%, which was the highest conversion rate of cellobiose to date. Therefore, this study presents a cost-effective and efficient method to enhance the conversion rate of cellobiose during the glycosylation process.