posted on 2024-02-29, 09:30authored byChenchen Li, Mengli Li, Miaomiao Hu, Ming Miao, Tao Zhang
3′-Sialyllactose (3′-SL) is among the foremost
and
simplest sialylated breast milk oligosaccharides. In this study, an
engineered Escherichia coli for high-titer
3′-SL biosynthesis was developed by introducing a multilevel
metabolic engineering strategy, including (1) the introduction of
precursor CMP-Neu5Ac synthesis pathway and high-performance α2,3-sialyltransferase
(α2,3-SiaT) genes into strain BZ to achieve de novo synthesis of 3′-SL; (2) optimizing the expression of glmS-glmM-glmU involved
in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathways, and constructing
a glutamine cycle system, balancing the precursor pools; (3) analysis
of critical intermediates and inactivation of competitive pathway
genes to redirect carbon flux to 3′-SL biosynthesis; and (4)
enhanced catalytic performance of rate-limiting enzyme α2,3-SiaT
by RBS screening, protein tag cloning. The final strain BZAPKA14 yielded
9.04 g/L 3′-SL in a shake flask. In a 3 L bioreactor, fed-batch
fermentation generated 44.2 g/L 3′-SL, with an overall yield
and lactose conversion of 0.53 g/(L h) and 0.55 mol 3′-SL/mol,
respectively.