| Pyrroloquinoline quinone(PQQ) is the coenzyme of D-sorbitol dehydrogenase or L-sorbose dehydrogenase and L-sorbosone dehydrogenase in Gluconobacter oxydans, and Ketogulonicigenium vulgare, respectively In previous studies, a G. oxydans recombinant strain was constructed for the direct production of 2-Keto-L-gulonic acid(2-KLG), which was an important precursor of vitamin C. However, it was demonstrated that the inefficient biosynthesis and regeneration of PQQ limited the production of 2-KLG. In this study, G. oxydans WSH-003 was chosen to study the PQQ biosynthesis pathway. The platform including a high- throughput PQQ measurement, electroporation of G. oxydans and a system for marker- free gene deletion in G. oxydans, which were necessary for investigating the biosynthesis and regeneration of PQQ, were constructed. Based on this platform, the impact of overexpression of each PQQ biosynthetic genes or deletion of tld D gene on the production of PQQ, D-sorbitol dehydrogenase activity and growth rate of G. oxydans was discussed. Main results were described as following:(1) The D-glucose dehydrogenase(PQQGDH) was purified and a high-throughput PQQ measurement was constructed. The PQQGDH form Escherichia coli K-12 was expressed in E. coli BL21(DE3). The PQQGDH was p urified by nickel-affinity chromatography column. The volume of PQQGDH and PQQ sample used in the high-throughput PQQ measurement process was determined by ortho gonal experiment. The result demonstrated that the reaction system, which consisted of 30 μL PQQGDH, 2 μL PQQ sample and 1.2 m L chromogenic reagent, showed the best performence. The high- throughput measurement of PQQ using purified PQQGDH, 96-well plate and microplate reader are more accurate and efficient than traditional methods. The results obtained here would pay the way for measuring PQQ of culture medium in large-scale.(2) The electroporation method and a system for marker-free gene deletion were constructed in G. oxydans. G. oxydans of different growth period were choosen to prepare G. oxydans competent cells. Further, the effect of different electroporation conditions and post-cultural conditions on efficiency of electroporation were investigated. The result showed that G. oxydans which grow to an O D600 of 1.6-1.8 are best for preparing G. oxydans competent cells. Cells under the electroporation conditions including 0.1 cm cuvette, 1.8 k V voltage, 200 Ω resistance and 25 mF capacitance, 2 h post-culture performed best. An upp mutant named G.oxydans WSH-003 Δupp was constructed. The gene-deletion vector p K19 mobupp was derived from the vector p K19 mobsac B.,Marker- free gene deletion in G. oxydans was achieved by using G.oxydans WSH-003 Δupp and p K19 mobupp.(3) Overexpression of each PQQ biosynthetic genes and gene deletion of tld D was achieved in G. oxydans WSH-003. Six PQQ biosynthetic genes were overexpressed by two promoters in G. oxydans WSH-003, respectively. The result showed that overexpression of each single gene could enhance PQQ biosynthesis except for the tld D gene. In addition, the results show that PQQ levels were positively correlated with the efficiency of conversion of D-sorbitol to L-sorbose. This demonstrated that cofactor engineering of PQQ in G. oxydans was beneficial for enhancing the production of quinoprotein-related products. A tld D mutant G.oxydans was constructed successfully. The result showed that G.oxydans WSH-003 Δupp Δtld D growed slowly and was unable to synthesize PQQ. This further confirmed that the tld D was essential in PQQ biosynthesis and PQQ was beneficial for the growth of G.oxydans cel s. |
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