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Expectations for Employing Escherichia coli BL21 (DE3) in the Synthesis of Human Milk Oligosaccharides
Human milk oligosaccharides (HMOs) are unique components in breast milk, and their content in breast milk is second only to water, lactose, and fat. HMOs have aroused considerable commercial attention due to their unique effects on infant health. In addition, several HMOs, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), 3′-sialyllactose (3′-SL), and 6′-sialyllactose (6′-SL), have been approved by the Food and Drug Administration as “generally recognized as safe” foods that can be added to infant formula. (1) Currently, there are four methods for obtaining HMOs, which include separation from human milk, chemical synthesis, enzymatic synthesis, and microbial production. Although HMOs can be isolated from human milk through various purification steps, the relatively low yield is not conducive to the development of commercial application. The chemical synthesis requires multistep reactions for protection and deprotection procedures, making large-scale production of HMOs challenging. Enzymatic synthesis of HMOs requires expensive substrates and enzymes, which increases production costs. Microbial production uses cheap carbon and nitrogen sources as substrates to produce HMOs. The entire synthesis process is carried out in a constructed cell factory, which is easy to operate. Therefore, microbial production is the most promising option for producing HMOs. Here, we describe the application, challenges, and prospects for employing Escherichia coli BL21 (DE3) in the synthesis of HMOs (Figure 1). Figure 1. Application, challenges, and prospects for employing E. coli BL21 (DE3) in the synthesis of human milk oligosaccharides. E. coli has been used as the host for industrial production because of its easy cultivation, rapid reproduction, and clear genetic background. E. coli K-12 and B strains have been the subjects of classical experiments. E. coli BL21 (DE3), commonly used in the laboratory, was created by integrating the DE3 prophage into the λ attachment site of E. coli BL21 (B strain). The DE3 region of the prophage contains T7 RNA polymerase, which is a very active enzyme. It synthesizes RNA at a rate several times that of E. coli RNA polymerase. For this reason, the ability of E. coli BL21 (DE3) to express cloned genes is robust. (2) Learning from the success stories of the past decades, researchers have developed a variety of HMOs synthesis pathways based on the metabolic engineering of E. coli BL21 (DE3). Additionally, the European Commission Implementing Regulation approved the use of E. coli BL21 (DE3) in the production of HMOs (EU, 2017/2201; EU, 2015/2283). The development of HMO biosynthesis strains is routinely performed through the design–build–test–learn (DBTL) cycle. The first step in using E. coli BL21 (DE3) to produce HMOs is the identification of a metabolic pathway. The second step is the design of the metabolic pathway for the conversion of precursor molecules into HMOs. Next, the metabolic pathway is built using genetic engineering tools. Finally, the level of the transformation of precursor molecules into HMOs in E. coli BL21 (DE3) was tested through plasmid expression or chromosomal integration expression pathway genes. Once the production of HMOs is realized in E. coli BL21 (DE3), a cycle of DBTL is completed. (3) Over the past several decades, the construction of host strains for the synthesis of HMOs relied on random mutagenesis and overexpression of a single biosynthetic gene for the design and test steps. Although this method is widely used to improve the production of HMOs, it is often time-consuming and labor-intensive. Along with the development of synthetic biology tools, the establishment of metabolic flux analysis, genome-scale metabolic models and metabonomics analysis, and other related tools can predict more genetic modifications, which may lead to the acceleration of the engineering of E. coli BL21 (DE3). In recent years, plasmid expression systems have been used in engineering E. coli BL21 (DE3) for the production of HMOs, which has led to continuous antibiotic and gene expression instability problems. In the coming years, the target genes should be integrated into the chromosome to build a plasmid-free expression system. In addition, the complex and branched HMOs account for a large proportion of the total HMOs, while there have been few studies on the production of branched and complex HMOs by E. coli BL21 (DE3). (3,4) In the future, a low-cost microbial cell factory should be developed through modular engineering for the highly efficient production of complex and branched HMOs. E. coli Nissle 1917 (O6: K5: H1), a special E. coli strain, is nonpathogenic and is used as a probiotic in medicine and to treat various gastrointestinal diseases. It was originally isolated from the feces of a healthy person by A. Nissle in 1917. For nearly 100 years, E. coli Nissle 1917 has been the active pharmaceutical ingredient of the licensed drug Mutaflor that is distributed on the drug market in Germany and several other countries. For this reason, E. coli Nissle 1917 has recently been applied in synthetic biology. (5) Engineering of E. coli Nissle 1917 employed for the production of HMOs can be further developed in the future. This probiotic expression system used to produce HMOs will be competitive in the commercial market. In conclusion, HMOs are complex carbohydrates that are abundant in human milk and are known to play important roles in infant health and development. Thus, there is increasing interest in developing cell factories capable of synthesizing HMOs in a cost-effective and sustainable manner. Although there are still challenges in achieving the efficient and cost-effective production of branched and complex HMOs, the prospect of the synthesis of HMOs in cell factories is promising, as it could provide a sustainable and scalable source of HMOs for use in infant formula and other applications. In the coming years, further research is needed to optimize the production of HMOs in cell factories and to fully understand the potential benefits and limitations of improving infant nutrition and health with HMOs. This research was financially supported by the Major Science and Technology Innovation Project of Shandong Province (2020CXGC010601). This article references 5 other publications. This article has not yet been cited by other publications. Figure 1. Application, challenges, and prospects for employing E. coli BL21 (DE3) in the synthesis of human milk oligosaccharides. This article references 5 other publications.
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