National Glycoengineering Research Center, Shandong University, Qingdao, People's Republic of China.
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, People's Republic of China.
Appl Environ Microbiol. 2019 Oct 16;85(21). doi: 10.1128/AEM.01367-19. Print 2019 Nov 1.
is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of 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 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 WCFS1 toward producing -acetylglucosamine (GlcNAc). was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in 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. 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 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. WCFS1 produced 797.3 mg/liter -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.
是一种潜在的起始和促进健康的益生菌细菌。有效地、精确地和多样化地编辑 ，而不引入外源基因或质粒，这是非常重要的。在本研究中，在 WCFS1 中建立了 CRISPR/Cas9 辅助的双链 DNA（dsDNA）和单链 DNA（ssDNA）重组，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，使用硫代磷酸酯修饰来提高 dsDNA 插入效率，并使用腺嘌呤特异性甲基转移酶来提高 ssDNA 重组效率。这些策略被应用于工程化 ，以生产 -乙酰葡萄糖胺（GlcNAc）。截短 以消除果糖-6-磷酸（F6P）到葡萄糖胺 6-磷酸（GlcN-6P）的逆向反应。引入 核糖开关替换和点突变以缓解反馈抑制。产生的菌株在不引入外源基因或质粒的情况下生产了 797.3mg/l 的 GlcNAc。该策略可能有助于提供用于乳酸菌精确和多样化遗传工程的方法，并推动用于更多应用的菌株工程。CRISPR/Cas9 辅助的重组在乳酸菌中受到限制，因为缺乏可用的抗生素和载体。在本研究中，在 中使用 CRISPR/Cas9 辅助的双链 DNA（dsDNA）和单链 DNA（ssDNA）重组进行了无缝基因组编辑，通过内源腺嘌呤特异性甲基转移酶的过表达有效地提高了重组效率。通过强化 GlcNAc 途径， 生产了 797.3mg/l 的 -乙酰葡萄糖胺（GlcNAc），而不引入外源基因或质粒。这种无缝编辑策略，结合潜在的外源 GlcNAc 产生途径，使该菌株成为未来工业应用的有吸引力的候选菌株。
