Vol.4 No.4 2012

44/62

Research paper : Efficient production of active form of vitamin D3 by microbial conversion (Y. Yasutake et al.)−234−Synthesiology - English edition Vol.4 No.4 (2012) AuthorsYoshiaki YasutakeCompleted the doctorate course at the Graduate School of Science, Hokkaido University in 2004. Industry-academia-government collaboration researcher, Graduate School of Science, Hokkaido University. Doctor (Science). Researcher, Research Institute of Genome-based Biofactory, AIST in April 2005. Researcher, Bioproduction Research Institute, AIST in April 2010. Currently engages in the researches for the VD3 hydroxylation reaction described in this paper, as well as the biosynthesis enzyme of new antibiotic material and the structural function analysis and achievement of high function for useful function proteins such as the medical diagnostic enzymes. In this paper, was in charge of the structural research, functional analysis, and functional alteration of the proteins involved in microbial conversion.Tomohiro TamuraCompleted the courses at the Faculty of Medicine, The University of Tokushima in 1993. Research Fellow of the Japan Society for the Promotion of Science. Doctor (Medicine). Post-doctorate at the Research Unit for Molecular Structural Biology, Max Planck Institute of Biochemistry in 1994. Joined the Hokkaido National Industrial Research Institute, Agency of Industrial Science and Technology (currently, AIST) in 2000. Professor of the Graduate School of Agriculture, Hokkaido University in 2002. Leader of Proteolysis and Protein Turnover Research Group, Bioproduction Research Institute, AIST in 2011. Visiting professor of the Graduate School of Agriculture, Hokkaido University. Currently engages in the development of expression platform that allows the use of the Rhodococcus actinomycetes for diverse purposes. In this paper, planned and summarized the research for overall VD3 hydroxylation reaction.Discussion with Reviewers1 Overall commentComment (Kazunori Nakamura, Biomedical Research Institute, AIST)Overall, the specific reaction system is not clearly stated in the paper. Also, since you do not state the specific figures for the cost and efficiency of the conventional method, it is difficult to determine quantitatively how much increased productivity and cost reduction were achieved in this research.Answer (Yoshiaki Yasutake, Tomohiro Tamura)I added the explanation pertaining to the reaction system as you indicated. However, this reaction system is a technology that is currently being used in production, and is a corporate secret. Therefore, I cannot disclose any specific information including the production efficiency. I added and modified the descriptions to the extent that I can disclose at this point. 2 Description of specific production methodComment (Kazunori Nakamura)In the description relating to bioconversion rate, you say that the issues are the slow rate of growth of the microorganisms and the reaction speed of the enzymes, but it is difficult to know which phase is a more serious problem since you do not describe the specific production method. For example, do you convert using non-growing cells after culturing the microorganisms, or using growing microorganisms? Please describe the specific production method in detail. Answer (Yoshiaki Yasutake, Tomohiro Tamura)As mentioned in Discussion 1, the various information relating to the current hydroxylated VD3 production system cannot be disclosed. Therefore, please understand that I cannot provide comparisons using actual figures. I added and modified the descriptions to the extent that I can disclose at this point.3 Future issues in realizationQuestion (Motoyuki Akamatsu, Human Technology Research Institute, AIST)You write that you developed a production method with extremely high production efficiency in this research, and you aim for further efficiency of electron transfer in “4. Future developments and issues”. Why do you need further efficiency? Pertaining to this, you mention that this is a joint research with Micro Biopharm Japan Co., Ltd. Do you need further R&D for actual production? Is the electron transfer efficiency the barrier for the production? Please describe any other barriers, if any, to clarify the positioning of the results.Answer (Yoshiaki Yasutake, Tomohiro Tamura)This R&D is an effort to enhance the efficiency and refinement of the production method using P. autotrophica that has already been realized. The productivity has greatly improved through the modification of the enzymes based on evolutionary engineering and 3D structure, as well as changes in the organic species. However, the thermostability of the enzyme with increased activity by the introduction of variation is low, and the issue that the enzyme cannot be accumulated in large amounts in the cell became apparent. Therefore, as a method for increasing activity while maintaining enzyme stability, we think the increased efficiency of electron transfer for P450 is necessary, and we infer that there are rooms for improvement in the Vdh, AciB, and C systems. By advancing the electron transfer efficiency, I think it is possible to go beyond the current production efficiency. On the other hand, the barrier in using the reaction system by R. erythropolis in actual production is not a matter of production efficiency, but is the problem of using recombinant bacteria. Currently, the company uses the breeding strain and does not engage in production by recombinant bacteria. To change the bacteria type and to start up the production system using the recombinant bacteria, various procedures including safety tests will become necessary, as well as additional facility investment to build such a plant. The market for active form of VD3 is expected to grow further, and I expect the production using the technology we developed will be in demand in the future. I modified the paper according to your comment.[12]H. E. Hasper, B. Kruijff and E. Breukink: Assembly and stability of nisin-lipid II pores, Biochemistry, 43 (36), 11567- 11575 (2004).[13]L. S. Koo, C. E. Immoos, M. S. Cohen, P. J. Farmer and P. R. Ortiz de Montellano: Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119, J. Am. Chem. Soc., 124 (20), 5684-5691 (2002).

※このページを正しく表示するにはFlashPlayer9以上が必要です