초록
<P><B>Abstract</B></P> <P>Chondroitin sulfates, widely used in the treatment of arthritis, are glycosaminoglycans extracted from food animal tissues. As part of our ongoing efforts to separate the food chain from the drug chain, we are examining the possibility of using metabolic engineering to produce chondroitin sulfate in <I>Escherichia coli</I>. Chondroitin is a valuable precursor in the synthesis of chondroitin sulfate. This study proposes a safer and more feasible approach to metabolically engineer chondroitin production by expressing genes from the pathogenic <I>E. coli</I> K4 strain, which natively produces a capsular polysaccharide that shares the similar structure with chondroitin, into the non-pathogenic <I>E. coli</I> BL21 Star™ (DE3) strain. The ePathBrick vectors, allowing for multiple gene addition and expression regulatory signal control, are used for metabolic balancing needed to obtain the maximum potential yield. The resulting engineered strain produced chondroitin, as demonstrated by <SUP>1</SUP>H NMR and disaccharide analysis, relying on chondrotinase treatment followed by liquid chromatography–mass spectrometry. The highest yield from shake flask experiment was 213mg/L and further increased to 2.4g/L in dissolved oxygen-stat fed batch bioreactor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Microbial production of chondroitin, an important nutraceutical glycosaminoglycan. </LI> <LI> Metabolic pathway balancing of related capsular polysaccharide biosynthetic pathways for optimal production. </LI> <LI> Development of an accurate analytically method for chondroitin quantification using LC–MS. </LI> <LI> Explore the feasibility of a scaled-up chondroitin production process. </LI> <LI> High product yield using a safer, non-pathogenic bacterial strain. </LI> </UL> </P>