Simonsiella muelleri α2,6-sialyltransferase enables efficient biosynthesis of 6′-sialyllactose in both plasmid-dependent and plasmid-free engineered Escherichia coli

IF 8.5 1区 化学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Yuanlin Liu , Yingying Zhu , Liping Qiao , Lumeng Yu , Qian Lin , Wei Xu , Wenli Zhang , Wanmeng Mu
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Abstract

6′-Sialyllactose (6′-SL) is the most abundant sialylated human milk oligosaccharide in breast milk. Its health effects have been widely confirmed, and its biological production attracts increasing attention. The metabolic pathway of 6′-SL synthesis is clear, and the typical metabolic engineering strategies for its production improvement have been reported. However, there are not many α2,6-sialyltransferase (α2,6-SiaT) candidates reported. In this study, α2,6-SiaT from Simonsiella muelleri ATCC 29453 was reported to be effective in vivo 6′-SL productivity in engineered Escherichia coli. Based on this α2,6-SiaT, a two-plasmid E. coli BL21(DE3) was constructed to produce 6′-SL. After plasmid combination optimization and RBS engineering, which optimizes gene expression and enhances target product production by designing and modifying ribosome binding site (RBS) sequences, the titer was enhanced to 3.405 and 21.020 g/L in shake-flask and fed-batch cultivation, respectively. In addition, E. coli MG1655 was engineered to produce 6′-SL by genomic multiple copy integration of pathway genes, and the fed-batch cultivation generated 17.220 g/L of 6′-SL without the use of a plasmid. This study provided a new α2,6-SiaT candidate for microbial synthesis of 6′-SL.
穆勒Simonsiella muelleri α2,6-唾液基转移酶能够在依赖质粒和不依赖质粒的工程大肠杆菌中高效地生物合成6 ' -唾液基乳糖
6 ' -唾液乳糖(6 ' -SL)是母乳中含量最多的唾液化人乳低聚糖。其保健作用已得到广泛证实,其生物生产日益引起人们的关注。6 ' -SL合成的代谢途径是明确的,并报道了典型的代谢工程策略来提高其产量。然而,α2,6-唾液基转移酶(α2,6- siat)候选物报道不多。在本研究中,报道了来自穆勒simonella muelleri ATCC 29453的α2,6- siat在工程大肠杆菌中具有有效的体内6 ' -SL产量。在α2,6- siat的基础上,构建了双质粒大肠杆菌BL21(DE3),生成了6′-SL。经质粒组合优化和通过设计和修饰核糖体结合位点(RBS)序列来优化基因表达和提高目标产物产量的RBS工程后,摇瓶培养和补料分批培养的滴度分别提高到3.405和21.020 g/L。此外,通过途径基因的基因组多拷贝整合,设计大肠杆菌MG1655产生6’-SL,在不使用质粒的情况下,分批补料培养产生了17.220 g/L的6’-SL。该研究为微生物合成6′-SL提供了新的α2,6- siat候选物。
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来源期刊
International Journal of Biological Macromolecules
International Journal of Biological Macromolecules 生物-生化与分子生物学
CiteScore
13.70
自引率
9.80%
发文量
2728
审稿时长
64 days
期刊介绍: The International Journal of Biological Macromolecules is a well-established international journal dedicated to research on the chemical and biological aspects of natural macromolecules. Focusing on proteins, macromolecular carbohydrates, glycoproteins, proteoglycans, lignins, biological poly-acids, and nucleic acids, the journal presents the latest findings in molecular structure, properties, biological activities, interactions, modifications, and functional properties. Papers must offer new and novel insights, encompassing related model systems, structural conformational studies, theoretical developments, and analytical techniques. Each paper is required to primarily focus on at least one named biological macromolecule, reflected in the title, abstract, and text.
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