Abstract
Objectives
Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium.
Results
The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated.
Conclusions
These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.
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Data availability
Strains and plasmids generated in this study will be made available upon request.
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This work was supported by the National Natural Science Foundation of China (31670120).
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XL and YY performed the experimental work; JT and BG wrote the paper; WL and TC conducted the NMR and MS analytics and interpretation of spectra; XZ conceived the study and secured the funding. All authors read and approved the manuscript.
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Li, X., Yu, Y., Tang, J. et al. The construction of a dual-functional strain that produces both polysaccharides and sulfotransferases. Biotechnol Lett 43, 1831–1844 (2021). https://doi.org/10.1007/s10529-021-03156-4
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DOI: https://doi.org/10.1007/s10529-021-03156-4