Elimination of Residual Lacto-N-triose II for Lacto-N-tetraose Biosynthesis in Engineered Escherichia coli.
Elimination of Residual Lacto-N-triose II for Lacto-N-tetraose Biosynthesis in Engineered Escherichia coli.
复制标题
消除工程大肠杆菌中乳-N-四糖生物合成中残留的乳-N-三糖 II。
DOI：
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发表时间：
2023
影响因子：
6.1
通讯作者：
W. Mu
中科院分区：
文献类型：
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作者：
Longhao Yang;Yingying Zhu;Chunhua Zhao;Mingli Zhao;Mengting Tao;Zeyu Li;Wenli Zhang;W. Mu
乳酸- n -四糖（LNT）是一种重要的中性人乳寡糖（HMO），是复杂HMO生物合成的重要核心结构。我们之前使用补料分批发酵实现了高产LNT生物合成（57.5 g/L）；而乳酸- n -三糖II （LNTri II）残留量为21.58 g/L。在这里，我们利用遗传稳定的LNTri II产生菌株重组了一种高效的低LNTri II积累的大肠杆菌，该菌株具有基因组插入lgtA（编码β1,3- n -乙酰氨基葡萄糖转移酶）。通过引入β1,3-半乳糖转移酶，获得了相当滴度的LNT （3.73 ~ 4.61 g/L）和低滴度的LNTri II （0.33 ~ 0.63 g/L）。为了减少LNTri II的残留，破坏了大肠杆菌转运体基因setA，明显减少了LNTri II和LNT的积累。接下来，将编码β- n -乙酰己糖氨酸酶（BbhI）的基因分别导入产生lnt的菌株和大肠杆菌BL21（DE3）中进行单菌种和混合菌种培养。最后，在混合工程菌株共培养体系中，获得了LNT (30.13 g/L)，没有多余的LNTri II。
Lacto-N-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-N-triose II (LNTri II) was also found (21.58 g/L). Here, we re-engineered an efficient LNT-producing Escherichia coli with low LNTri II accumulation using genetically stable LNTri II-producing strains with a genomic insertion of lgtA (encoding β1,3-N-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 E. coli transporter gene setA was disrupted, obviously reducing the accumulation of LNTri II and LNT. Next, the gene encoding β-N-acetylhexosaminidase (BbhI) was introduced into LNT-producing strains or E. coli 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.