Abstract
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 (104–105 CFUs/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 in E. coli by 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 of Lactococcus lactis kivD and Saccharomyces 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.
Highlights
DNA assembly is an essential technique enabling metabolic engineering and synthetic biology
The PS-Brick presents a valuable addition to the current toolbox of synthetic biology and metabolic engineering
Design and proof‐of‐concept of PS‐Brick assembly scheme The design principles of PS‐Brick Compared to the original BioBrick standard and Golden Gate assembly, PS-Brick applies both of them in the assembly reaction (Fig. 1a)
Summary
DNA 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. Current challenges of metabolic engineering focus on developing cell factories for specific metabolites that can be produced with titres, yields and productivities as high as possible for industrial application [1,2,3]. It is necessary to perform many rounds of proof-of-concept studies that first implement specific metabolic designs and generate new knowledge for improved design. In each DBTL cycle of metabolic engineering, enzyme screening, chimeric pathway reconstruction, fine-tuning optimization or metabolic bottleneck elimination is performed one by one, and the titre, yield or productivity of the desired metabolites for engineered strains is stepwise improved to high levels
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