Comparison of Multiple Gene Assembly Methods for Metabolic Engineering

Abstract

A universal, rapid DNA assembly method for efficient multigene plasmid construction is important for biological research and for optimizing gene expression in industrial microbes. Three different approaches to achieve this goal were evaluated. These included creating long complementary extensions using a uracil-DNA glycosylase technique, overlap extension polymerase chain reaction, and a SfiI-based ligation method. SfiI ligation was the only successful approach for assembling large DNA fragments that contained repeated homologous regions. In addition, the SfiI method has been improved over a similar, previous published technique so that it is more flexible and does not require polymerase chain reaction to incorporate adaptors. In the present study, Saccharomyces cerevisiae genes TAL1, TKL1, and PYK1 under control of the 6-phosphogluconate dehydrogenase promoter were successfully ligated together using multiple unique SfiI restriction sites. The desired construct was obtained 65% of the time during vector construction using four-piece ligations. The SfiI method consists of three steps: first a SfiI linker vector is constructed, whose multiple cloning site is flanked by two three-base linkers matching the neighboring SfiI linkers on Sfi I digestion; second, the linkers are attached to the desired genes by cloning them into SfiI linker vectors; third, the genes flanked by the threebase linkers, are released by SfiI digestion. In the final step, genes of interest are joined together in a simple one-step ligation.

Increasing cloning possibilities using artificial zinc finger nucleases

“…Thus, although other molecular techniques, such as overlap extension polymerase chain reaction (PCR), SfiI-based ligation, multiGATEWAY recombination, and uracil-DNA glycosylase, can be used for the construction of complex DNA structures (21,28), the search continues for novel and rare-cutting restriction enzymes that can facilitate the cloning of DNA molecules. Various approaches have been taken to creating artificial rare cutters, most involving engineering existing enzymes for unique specificities.…”

Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain

“…To create each of the library's individual cassettes (i.e., GND2-TAL1-HXK2-TKL1-HXK2-PYK1), three fusion products containing the above three genes were concatenated by using modified SfiI cleavage methods (23,37). Briefly, degenerate SfiI restriction sites were attached to each fusion product, and three base sticky ends were generated by SfiI cleavage of each construct and further assembled together into pSfi-314 in a one-step ligation procedure (23). Each assembled vector was transformed to parental strain FPLYSX3 by using the standard lithium acetate method, and the control strain, FPL314, was just FPLYSX3-transformed with empty vector pSfi-314.…”

“…Located in the lower glycolytic pathway, PYK1 determines the glycolytic rate and direction (27). Vectors consisting of eight promoter and gene combinations (see Table S1 in the supplemental material) were constructed using the method based on the SfiI degenerate recognition sequence (23). Enzyme assays and quantitative PCR FIG.…”