A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production
A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production
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一种名为 PS-Brick 的无缝迭代 DNA 组装方法及其用于苏氨酸和 1-丙醇生产的辅助代谢工程
DOI：
10.1186/s13068-019-1520-x
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发表时间：
2019-07-15
影响因子：
6.3
通讯作者：
Wen, Tingyi
中科院分区：
文献类型：
--
作者：
Liu, Shuwen;Xiao, Haihan;Wen, Tingyi
背景DNA组装是实现代谢工程和合成生物学的关键技术。将新型DNA组装技术与合理的代谢工程相结合，可以促进微生物细胞工厂的构建。氨基酸及其衍生物是工业生物技术中的重要产品，应用广泛，市场巨大。在代谢工程中遇到的DNA组装方案的氨基酸和相关化合物的生产者，如设计-构建-测试-学习周期，精确的遗传电路和重复的DNA分子的建设，通常需要迭代，无疤痕和重复序列组装方法，respectively.ResultsRestriction endonuclease（RE）-assisted strategies构成的DNA组装的主要类别之一。在这里，我们开发了一种称为PS-Brick的IIP型和IIS型RE辅助方法，该方法综合利用PCR片段和RE的特性进行迭代，无缝和重复的序列组装。使用纯化的质粒和PCR片段的一轮PS-Brick反应在几小时内完成，并且从该PS-Brick组装反应得到的反应产物的转化表现出高效率（104- 105 CFU/μg DNA）和高准确性（~ 90%）。代谢工程在苏氨酸生产中的应用，包括反馈调节的释放、代谢瓶颈的消除、苏氨酸输出的强化和苏氨酸催化剂的失活，在E. coliby轮的“设计-构建-测试-学习”循环，通过补料分批发酵获得45.71 g/L苏氨酸。除了PS-Brick的迭代特性对于序列菌株工程的价值之外，无缝克隆还能够实现密码子饱和诱变和双顺反子设计的精确框内融合，并且PS-Brick的重复序列克隆能力能够构建用于基因组编辑的串联CRISPR sgRNA阵列。此外，由乳酸乳球菌KivDan和酿酒酵母ADH 2组成的苏氨酸衍生1-丙醇途径的异源途径通过一个循环的PS-Brick组装，在流加发酵中产生1.35 g/L的1-丙醇。结论据我们所知，PS-Brick框架是第一个结合IIP型和IIS型RE的优点的RE辅助DNA组装方法。PS-Brick被证明是一种用于途径构建和基因组编辑的有效DNA组装方法，并成功应用于代谢工程的设计-构建-测试-学习（DBTL）循环，用于生产苏氨酸和苏氨酸衍生的1-丙醇。PS-Brick为合成生物学和代谢工程的现有工具箱提供了有价值的补充。
BackgroundDNA assembly is an essential technique enabling metabolic engineering and synthetic biology. Combining novel DNA assembly technologies with rational metabolic engineering can facilitate the construction of microbial cell factories. Amino acids and derived biochemicals are important products in industrial biotechnology with wide application and huge markets. DNA assembly scenarios encountered in metabolic engineering for the construction of amino acid and related compound producers, such as design-build-test-learn cycles, construction of precise genetic circuits and repetitive DNA molecules, usually require for iterative, scarless and repetitive sequence assembly methods, respectively.ResultsRestriction endonuclease (RE)-assisted strategies constitute one of the major categories of DNA assembly. Here, we developed a Type IIP and IIS RE-assisted method named PS-Brick that comprehensively takes advantage of the properties of PCR fragments and REs for iterative, seamless and repetitive sequence assembly. One round of PS-Brick reaction using purified plasmids and PCR fragments was accomplished within several hours, and transformation of the resultant reaction product from this PS-Brick assembly reaction exhibited high efficiency (104–105CFUs/µg DNA) and high accuracy (~ 90%). An application of metabolic engineering to threonine production, including the release of feedback regulation, elimination of metabolic bottlenecks, intensification of threonine export and inactivation of threonine catabolism, was stepwise resolved inE. coliby rounds of “design-build-test-learn” cycles through the iterative PS-Brick paradigm, and 45.71 g/L threonine was obtained through fed-batch fermentation. In addition to the value of the iterative character of PS-Brick for sequential strain engineering, seamless cloning enabled precise in-frame fusion for codon saturation mutagenesis and bicistronic design, and the repetitive sequence cloning ability of PS-Brick enabled construction of tandem CRISPR sgRNA arrays for genome editing. Moreover, the heterologous pathway deriving 1-propanol pathway from threonine, composed ofLactococcus lactis kivDandSaccharomyces cerevisiae ADH2, was assembled by one cycle of PS-Brick, resulting in 1.35 g/L 1-propanol in fed-batch fermentation.ConclusionsTo the best of our knowledge, the PS-Brick framework is the first RE-assisted DNA assembly method using the strengths of both Type IIP and IIS REs. In this study, PS-Brick was demonstrated to be an efficient DNA assembly method for pathway construction and genome editing and was successfully applied in design-build-test-learn (DBTL) cycles of metabolic engineering for the production of threonine and threonine-derived 1-propanol. The PS-Brick presents a valuable addition to the current toolbox of synthetic biology and metabolic engineering.