Simultaneous production of N-acetylheparosan and recombinant chondroitin using gene-engineered Escherichia coli K5. Issue 4 (April 2023)

• Record Type: Journal Article
• Authors: Suzuki, Kiyoshi; Kaseyama-Takemoto, Hiromi
• Is Part Of: Heliyon. Volume 9:Issue 4(2023)
• Publication Date: 2023-04
• Subjects: Recombinant E. coli K5 -- Batch culture -- Chondroitin -- N-Acetylheparosan, and co-production; Research -- Periodicals; Medical sciences -- Periodicals; Natural history -- Periodicals; Social sciences -- Periodicals; Earth sciences -- Periodicals; Physical sciences -- Periodicals; 507.2
• DOI: 10.1016/j.heliyon.2023.e14815
• Languages: English
• ISSNs: 2405-8440

Abstract

Abstract: N-Acetylheparosan and chondroitin are increasingly needed as alternative sources of animal-derived sulfated glycosaminoglycans (GAGs) and as inert substances in medical devices and pharmaceuticals. The N- acetylheparosan productivity of E. coli K5 has achieved levels of industrial applications, whereas E.coli K4 produces a relatively lower amount of fructosylated chondroitin. In this study, the K5 strain was gene-engineered to co-express K4-derived, chondroitin-synthetic genes, namely kfoA and kfoC . The productivities of total GAG and chondroitin in batch culture were 1.2 g/L and 1.0 g/L respectively, which were comparable to the productivity of N -acetylheparosan in the wild K5 strain (0.6–1.2 g/L). The total GAG of the recombinant K5 was partially purified by DEAE-cellulose chromatography and was subjected to degradation tests with specific GAG-degrading enzymes combined with HPLC and 1H NMR analyses. The results indicated that the recombinant K5 simultaneously produced both 100-kDa chondroitin and 45-kDa N - acetylheparosan at a weight ratio of approximately 4:1. The content of chondroitin in total GAG partially purified was 73.2%. The molecular weight of recombinant chondroitin (100 kDa) was 5–10 times higher than that of commercially available chondroitin sulfate. These results indicated that the recombinant K5 strain acquired the chondroitin-producing ability without altering the total GAG productivity of the host. Graphical abstract: Image 1 Highlights: E. coli K5 transformed with CH biosynthetic genes acquired CH-producing ability. This bacterium simultaneously produced both CH and HPN in a single fermentation. Digestion of the culture with specific enzymes helps to isolate CH or HPN singly. Recombinant CH (Mw: 100 kDa) was 5–10 times larger than ordinary CS.