State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China.
Bloomature Biotechnology Co., Ltd., Beijing 102629, China.
J Agric Food Chem. 2023 Aug 23;71(33):12511-12518. doi: 10.1021/acs.jafc.3c03644. Epub 2023 Aug 9.
Lacto--tetraose (LNT) is an important neutral human milk oligosaccharide (HMO) and acts as a significant core structure for complex HMO biosynthesis. We previously achieved high-yield LNT biosynthesis (57.5 g/L) using fed-batch fermentation; however, residual lacto--triose II (LNTri II) was also found (21.58 g/L). Here, we re-engineered an efficient LNTproducing with low LNTri II accumulation using genetically stable LNTri II-producing strains with a genomic insertion of (encoding β1,3--acetylglucosaminyltransferase). Comparable and low titers of LNT (3.73-4.61 g/L) and LNTri II (0.33-0.63 g/L), respectively, were obtained by introducing β1,3-galactosyltransferase. To reduce residual LNTri II, the transporter gene was disrupted, obviously reducing the accumulation of LNTri II and LNT. Next, the gene encoding β--acetylhexosaminidase () was introduced into LNT-producing strains or BL21(DE3) for single- or mixed-strain cultivation, respectively. Finally, LNT was obtained (30.13 g/L) in a cocultivation system of mixed engineered strains without undesired LNTri II.
乳四糖 (LNT) 是一种重要的中性人乳寡糖 (HMO)，作为复杂 HMO 生物合成的重要核心结构。我们之前通过分批补料发酵实现了高产 LNT 生物合成（57.5 g/L）；然而，也发现了残留的乳三糖 II (LNTri II)（21.58 g/L）。在这里，我们使用基因稳定的 LNTri II 生产菌株进行了基因改造，在基因组中插入了 （编码β1,3--乙酰氨基葡萄糖基转移酶），从而实现了低 LNTri II 积累的高效 LNT 生产。通过引入β1,3-半乳糖基转移酶，分别获得了可比的和低的 LNT（3.73-4.61 g/L）和 LNTri II（0.33-0.63 g/L）产量。为了减少残留的 LNTri II，破坏了 转运蛋白基因 ，明显减少了 LNTri II 和 LNT 的积累。接下来，分别将编码β--乙酰己糖胺酶 () 的基因导入 LNT 生产菌株或 BL21(DE3)，用于单菌株或混合菌株培养。最后，在混合工程菌株的共培养系统中获得了 LNT（30.13 g/L），而没有不期望的 LNTri II。
