Efficient Production of a Functional Human Milk Oligosaccharide 3′-Sialyllactose in Genetically Engineered Escherichia coli

Abstract

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 α2,3-sialyltransferase-encoding gene were introduced into Escherichia coli BL21­(DE3)­ΔlacZ 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 α2,3-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 α2,3-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.

Efficient Production of N-Acetylneuraminic Acid in Escherichia coli Based on the UDP-N-Acetylglucosamine Biosynthetic Pathway

“…Interestingly, the engineered strains EM11 and EM12, in which neuC and neuB were separately inserted in the two MCS sites of pETDuet-1, produced relatively low levels of NeuAc. In previous studies, the location and mode of pathway gene insertion in the MCS sites could also affect the synthesis of target chemicals, such as 3′-sialyllactose . In addition, the lack of neuC (EM13) or neuB (EM14) failed to produce NeuAc, even if the UDP-GlcNAc pathway was strengthened (Figure ).…”

“…In addition, UDP-GlcNAc served as the direct precursor of ManNAc, and to enhance UDP-GlcNAc supply for ManNAc and NeuAc production, nagB and wecB were selected to be blocked. The deletion of nagB and/or wecB was proven to be beneficial to enhance the production of chemicals through the UDP-GlcNAc pathway, such as lacto- N -triose II, lacto- N -tetraose, lacto- N -neotetraose, 3′-sialyllactose, and also NeuAc . Herein, based on the strain EM5, additional deletion of nagB and nagB - wecB was performed to generate EM6 and EM7, respectively.…”

“…coli, UDP-GlcNAc is produced from Fru6P, the basic intermediate of the glycolytic pathway, by three consecutive enzymes including GlmS, GlmM, and GlmU (Figure ). In previous studies, the homologous overexpression of UDP-GlcNAc-producing enzymes significantly enhanced the metabolic engineering biosynthesis of chemicals with UDP-GlcNAc as the precursor, such as lacto- N -triose II, 3′-sialyllactose, and also NeuAc. The effect of strengthening various UDP-GlcNAc pathway genes was previously studied for NeuAc synthesis.…”

“…In previous studies, the location and mode of pathway gene insertion in the MCS sites could also affect the synthesis of target chemicals, such as 3′-sialyllactose. 30 In addition, the lack of neuC (EM13) or neuB (EM14) failed to produce NeuAc, even if the UDP-GlcNAc pathway was strengthened (Figure 3).…”

An Overview of Sugar Nucleotide-Dependent Glycosyltransferases for Human Milk Oligosaccharide Synthesis

“…After optimization, 3′-SL was extracellularly generated with the titers of 4.5 and 23.1 g/L by shake-flask and fed-batch fermentation, respectively. 115 4.2. α2,6-SiaT. α2,6-SiaT was characterized for the first time from Photobacterium damselae subsp.…”