CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production
CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production
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CRISPR/Cas9辅助植物乳杆菌无缝基因组编辑及其在N-乙酰氨基葡萄糖生产中的应用
DOI：
10.1128/aem.01367-19
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发表时间：
2019
影响因子：
4.4
通讯作者：
Qi Qingsheng
中科院分区：
文献类型：
--
作者：
Zhou Ding;Jiang Zhennan;Pang Qingxiao;Zhu Yuan;Wang Qian;Qi Qingsheng
由于缺乏可用的抗生素和载体，CRISPR/Cas9 辅助重组工程在乳酸菌中受到限制。本研究利用CRISPR/Cas9辅助的双链DNA（dsDNA）和单链DNA（ssDNA）重组工程在植物乳杆菌中进行了无缝基因组编辑方法，并通过内源腺嘌呤特异性甲基转移酶的过表达有效提高了重组效率。植物乳杆菌 WCFS1 通过强化 GlcNAc 途径产生 797.3mg/L N-乙酰氨基葡萄糖 (GlcNAc)，无需引入外源基因或质粒。这种无缝编辑策略与潜在的外源 GlcNAc 生产途径相结合，使该菌株成为未来工业用途的有吸引力的候选者。摘要 植物乳杆菌是一种潜在的发酵剂和促进健康的益生菌。在不引入外源基因或质粒的情况下对植物乳杆菌进行有效、精确和多样化的基因组编辑具有重要意义。在这项研究中，在植物乳杆菌WCFS1中建立了CRISPR/Cas9辅助的双链DNA（dsDNA）和单链DNA（ssDNA）重组工程，以无缝编辑基因组，包括基因敲除、插入和点突变。为了优化我们的编辑方法，使用硫代磷酸修饰来改善 dsDNA 插入，并使用腺嘌呤特异性甲基转移酶来提高 ssDNA 重组效率。这些策略被应用于改造植物乳杆菌 WCFS1，以生产 N-乙酰氨基葡萄糖 (GlcNAc)。 nagB 被截短以消除 6-磷酸果糖 (F6P) 与 6-磷酸氨基葡萄糖 (GlcN-6P) 的逆反应。引入核糖开关替换和 glmS1 点突变来缓解反馈抑制。所得菌株在未引入外源基因或质粒的情况下产生797.3mg/升GlcNAc。该策略可能有助于为乳酸菌提供精确多样的基因工程方法，并促进菌株工程的更多应用。重要性 由于缺乏可用的抗生素和载体，CRISPR/Cas9 辅助重组工程在乳酸菌中受到限制。本研究利用CRISPR/Cas9辅助的双链DNA（dsDNA）和单链DNA（ssDNA）重组工程在植物乳杆菌中进行了无缝基因组编辑方法，并通过内源腺嘌呤特异性甲基转移酶的过表达有效提高了重组效率。植物乳杆菌 WCFS1 通过强化 GlcNAc 途径产生 797.3mg/L N-乙酰氨基葡萄糖 (GlcNAc)，无需引入外源基因或质粒。这种无缝编辑策略与潜在的外源 GlcNAc 生产途径相结合，使该菌株成为未来工业用途的有吸引力的候选者。
CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future. ABSTRACT Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications. IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.