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Research paper : Efficient production of active form of vitamin D3 by microbial conversion (Y. Yasutake et al.)−233−Synthesiology - English edition Vol.4 No.4 (2012) reaction system and to have a stable redox partner in the cell[10]. Glucose dehydroxylase (GDH) was used as the NADH regenerator, and the reaction system using the wild-type Vdh, in which the AciB and AciC derived from Acinetobacter were co-expressed, was constructed as a highly stable redox partner (Fig. 6). The hydroxylated VD3 productivity of the nisin-treated cell was observed using this system. As a result, it was confirmed that hydroxylation efficiency was several times higher in the nisin-treated cells, compared to that of the untreated cells. It was also found that when the reaction, where one cycle consisted of a 16-hour reaction, was repeated in the nisin-treated cell, the VD3 hydroxylation rate per cycle increased to maximum 90 % (less than 50 % in untreated cells), and the total yield of 25(OH)VD3 after four reaction cycles was about six times higher compared to the untreated cell[10]. The conversion reaction system using nisin could convert 90 % of the VD3 in a short time, and significantly increased the production efficiency of the 25(OH)VD3. Moreover, since the nisin-treated cell used the buffer system as the reaction solution instead of culture media, the amount of foreign substances could be reduced. Also, the cells could be recovered and reused, and this is an effective method in the case where the productivity must be raised by increasing the number of reactions using the substrate with low solubility. If this technology could be used in P. autotrophica, we believe we can create a production system where highly active enzyme Vdh-K1 can be maximized. 4 Future developments and issuesThis R&D was conducted to find an efficient production method of active form of VD3 by microbial conversion and to construct an excellent conversion system in terms of efficiency and cost by overcoming the issues of conversion by wild-type strains that are currently done by the companies. The conversion system of R. erythropolis cell treated with nisin that was constructed in this research may realize a production efficiency that surpasses the system using P. autotrophica. Currently, we are engaging in the investigation of whether further efficiency can be achieved in the electron transfer between the redox partner and P450. Since the structural stability of the highly active mutant (Vdh-K1) is reduced, we think it is necessary to construct a system that promotes activity while maintaining the thermostability. There are already reports that the electron transfer efficiency has been increased in P450 and the activity is increased[13], and the introduction of the mutation on the ferredoxin binding region is expected to increase the conversion performance. On the other hand, the substance conversion technology that combines CD and the formation of pores by nisin treatment can be applied widely to the conversion system of substances where the highly hydrophobic, poorly soluble substances and CD are used as carriers. In general, the achievement of high efficiency in the microbial conversion of fat-soluble substance is very difficult, and we think there is high value in such usage. In the future, we would like to evaluate the usage value of nisin and CD in other microbial conversion systems. AcknowledgementsThis research was conducted jointly with the Pharmaceutical Chemical Division, Mercian Corporation (currently, this division is part of Micro Biopharm Japan Co., Ltd.), with the support of research funds from the NEDO Project “Development of Basic Technologies for Advanced Production Methods Using Microorganism Functions”. We express our sincere gratitude. References[1]G. Jones, S. A. Strugnell and H. F. DeLuka: Current understanding of the molecular actions of vitamin D, Physiol. Rev., 78 (4), 1193-1231 (1998).[2]G. D. Zhu and W. H. Okamura: Synthesis of vitamin D (calciferol), Chem. Rev., 95 (6), 1877-1952 (1995).[3]J. Sasaki, A. Miyazaki, M. Saito, T. Adachi, K. Mizoue, K. Hanada and S. Omura: Conversion of vitamin D3 to 1, 25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains, Appl. Microbiol. Biotechnol., 38 (2), 152-157 (1992).[4]K. Takeda, T. Asou, A. Matsuda, K. Kimura, K. Okamura, R. Okamoto, J. Sasaki, T. Adachi and S. 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Tamura: Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase, J. Biol. Chem., 285 (41), 31193-31201 (2010).[9]K. Nishimura, Y. Fujii, A. Arisawa, T. Tamura and Y. Yasutake: Improvement of vitamin D hydroxylase, Jpn. Kokai Tokkyo Koho JP 2011-115078 (June 16, 2011).[10]N. Imoto, T. Nishioka and T. Tamura: Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3 by Rhodococcus erythropolis, Biochem. Biophys. Res. Commun., 405 (3), 393-398 (2010).[11]W. Liu and J. N. Hansen: Some chemical and physical properties of nisin, a small protein antibiotic produced by Lactococcus lactis, Appl. Environ. Microbiol., 56 (8), 2551- 2558 (1990).

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