Cloning, Expression And Characterization Of Thermophilic Transaldolase And Transketolase And Their Preliminary Applications In In Vitro Synthetic Biology

In vitro synthetic biology as a branch of synthetic biology is an emerging area, which assembles catalytic building blocks in vitro to produce desired products through artificially designed synthetic pathways.In vitro synthetic biology possesses the advantages of high theoretical product yield, easy control of biosynthetic process and fewer byproducts, thus it has drawn the global attention for its great potential in application for production of drugs or drug precursors, food, biofuels and chemicals. Pathway design and catalytic element (enzyme) discovery are crucial to the in vitro synthetic biology application. Here, thermophilic enzymes are expected as promising catalytic elements for their remarkable thermostability and activity.Transaldolase and transketolase are the essential enzymes of the pentose phosphate pathway. They have already been used in the carbon-carbon coupling biotransformation, bio-hydrogen production and bio-fuel cells. Thermostable transaldolase and transketolase could be developed as catalytic elements for such applications. In this thesis work, the genes encoding transaldolase and transketolase were cloned from Thermotoga maritima and expressed in Escherichia coli BL21.The recombinant transaldolase and transketolase were found to be highly thermophilic with the optimal temperature of 80℃ and respective activities were 42 U/mg and 0.89 U/mg. They showed an outstanding thermostability:the half-life time of 198 h and 13 h at 60℃ and 80℃ for transaldolase and 365 h at 60℃ for transketolase. The estimated total turn-over numbers (TTNs) were 1.5×106 mol/mol and 1.2×103mol/mol for transaldolase and transketolase at the optimal reaction condition, which suggests their cost-effective application potential in in vitro synthetic biology.Furthermore, the application potential of the above-obtained thermophilic transaldolase and transketolase in in vitro synthetic biology was explored. Recently, the synthesis of sedoheptulose-7-phosphate and 2-epi-5-epi-valiolone has raised much interest as they are the important precursors of CyN-aminocyclitols such as the anti-diabetic agent acarbose and the crop protectant validamycin A. We designed the artificial synthetic pathways for sedoheptulose-7-phosphate and 2-epi-5-epi-valiolone by using the catalytic elements including the above-mentioned thermostable transaldolase and transketolase. In addition, the thermostable transaldolase was already applied to biohydrogen production from carbohydrates such as sucrose, fructose, glucose or xylose by our collaborator-Prof. YHP Zhang’s lab.This study suggests that these two highly active and thermostable enzymes could be promising building blocks for in vitro synthetic pathway biotransformation, and they are expected to have great potential in the synthesis of value-added (bio)chemicals, biofuels and so on.