title: Multiplex iterative plasmid engineering for combinatorial optimization of metabolic pathways and diversification of protein coding sequences.
abstract: Engineering complex biological systems typically requires combinatorial optimization to achieve the desired functionality. Here, we present Multiplex Iterative Plasmid Engineering (MIPE), which is a highly efficient and customized method for combinatorial diversification of plasmid sequences. MIPE exploits ssDNA mediated λ Red recombineering for the introduction of mutations, allowing it to target several sites simultaneously and generate libraries of up to 10(7) sequences in one reaction. We also describe "restriction digestion mediated co-selection (RD CoS)", which enables MIPE to produce enhanced recombineering efficiencies with greatly simplified coselection procedures. To demonstrate this approach, we applied MIPE to fine-tune gene expression level in the 5-gene riboflavin biosynthetic pathway and successfully isolated a clone with 2.67-fold improved production in less than a week. We further demonstrated the ability of MIPE for highly multiplexed diversification of protein coding sequence by simultaneously targeting 23 codons scattered along the 750 bp sequence. We anticipate this method to benefit the optimization of diverse biological systems in synthetic biology and metabolic engineering.
authors.affiliation: Key Laboratory of Systems Bioengineering, Ministry of Education, and Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University , Tianjin 300072, People's Republic of China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Li
authors.forename: Qun
authors.initials: Q
authors.lastname: Gu
authors.forename: Zhenquan
authors.initials: Z
authors.lastname: Lin
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.forename: Xueming
authors.initials: X
authors.lastname: Zhao
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C071147:ribB protein, E coli; C105392:RibC protein, bacteria; D017853:Phosphotransferases (Alcohol Group Acceptor); D009713:Nucleotidyltransferases; D019751:Intramolecular Transferases