Journal
ACS SYNTHETIC BIOLOGY
Volume 11, Issue 8, Pages 2837-2845Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.2c00243
Keywords
3 '-sialyllactose; human milk oligosaccharides; chromosomal integration; metabolic engineering; fed-batch cultivation
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Funding
- Key-Area Research and Development Program of China [2021YFD2100700]
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- DNA fragments used for construction of 3 ?
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In this study, a plasmid-based pathway optimization along with chromosomal integration strategies was used to successfully synthesize 3'-Sialyllactose (3'-SL). By deleting specific genes and optimizing key enzyme expression, the production of 3'-SL was significantly improved. The high yield achieved in large-scale fermentation is of great importance for the production of 3'-SL.
3 '-Sialyllactose (3 ' -SL) is one of the most important and simplest sialylated human milk oligosaccharides. In this study, a plasmid-based pathway optimization along with chromosomal integration strategies was applied for 3 '-SL production. Specifically, the precursor CMP-Neu5Ac synthesis pathway genes and alpha 2,3-sialyltransferase-encoding gene were introduced into Escherichia coli BL21-(DE3)delta lacZ to realize 3 ' -SL synthesis. Genes nanA and nanK involved in Neu5Ac catabolism were further deleted to reduce the metabolic flux of competitive pathway. Several alpha 2,3-sialyltransferases from different species were selected to evaluate the sialylation effect. The precursor pools were balanced and improved by optimizing key enzyme expression involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathway. Finally, an additional alpha 2,3-sialyltransferase expression cassette was integrated into chromosome to maximize 3 ' -SL synthesis, and 4.5 g/L extracellular 3 ' -SL was produced at a shake-flask level. The extracellular 3 ' -SL concentration was raised to 23.1 g/L in a 5 L bioreactor fermentation, which represents the highest extracellular value ever reported.
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