Biosynthesis of non-sulfated high-molecular-weight glycosaminoglycans and specific-sized oligosaccharides

Non-sulfated forms of glycosaminoglycans (NSGAGs) including hyaluronan, chondroitin and heparosan with high-molecular-weight (HMW) are extensively used biomaterials, while NSGAGs oligosaccharides display strong bioactivities. However, microbial production of HMW-NSGAGs and oligosaccharides with specific size are always challenging. Here, a membrane shield strategy was developed to produce HMW-NSGAGs by recruiting type II NSGAG synthases in Corynebacterium glutamicum. By enhancing precursor supplies and reinforcing cell membrane, the MWs of hyaluronan, heparosan and chondroitin reached 4100 kDa, 3000 kDa and 2400 kDa, respectively. In parallel, a synchronized depolymerization-polymerization strategy was developed by co-expressing NSGAGs synthases and lyases. With cell membrane as a filter, we achieved the direct biosynthesis of NSGAGs tetrasaccharides and disaccharides. The titers of chondroitin, hyaluronan and heparosan tetrasaccharides and disaccharides reached 10.9 g L−1, 12.1 g L−1 and 5.8 g L−1, respectively. The strategies developed here should also be applicable to the biosynthesis of other polysaccharides.