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Research paper : Efficient production of active form of vitamin D3 by microbial conversion (Y. Yasutake et al.)−228−Synthesiology - English edition Vol.4 No.4 (2012) these characteristics. However, the productivity has not been maximized since there are various issues that will be explained later. In this paper, the issues and the development goals of the microbial conversion that has been currently realized are presented. Then, the research methods used to solve the issues, how the methods were combined, and the ideas that turned out to be useful are explained to illustrate the construction of a highly efficient and high performing recombinant microbial conversion system.Please note that, in this paper, the information pertaining to the VD3 hydroxide production, which is currently being done commercially by pharmaceutical companies, cannot be disclosed, and we cannot present the actual figures for how much the developed technology contributed in increasing the efficiency of the production. 2 Issues that must be overcome and the development goalThe advantages of the activated VD3 production technology by microbial conversion are as stated above. Currently, the companies employ the method of culturing the breeding strain of P. autotrophica that has high VD3 hydroxylation capacity, adding the VD3 and cyclodextrin (CD) that increases the solubility of VD3, and accumulating the hydroxylated VD3 while growing the microorganisms (Fig. 1B). In the early research, the wild strain P. autotrophica was used, and 200 g/ml VD3 was added two days after culturing, and accumulation of 45 g/ml 25(OH)VD3 was observed after three days of culture[4]. Currently, the breeding strains are used for production, and the production efficiency is thought to have increased significantly.However, there are issues that must be improved in this method, and achievement of microbial conversion with dramatically increased performance can be expected by solving the issues. The issues and the expected causes are analyzed and summarized as a solution guideline as follows.(1)Conversion efficiency: In the current microbial conversion, not all VD3 added to the culture are converted, and there are remnant unreacted substrates. In the laboratory level analysis, over 30 % of unreacted substances are observed. Since the convertible amount may be dependent on the absolute amount of intracellular enzymes, the technology to stably express and accumulate a large amount of enzymes in the cell is necessary. By doing so, it may become possible to construct a conversion system where the majority of the added VD3 can be converted into the active form.(2)Conversion rate: In the currently conducted microbial conversion, over 100 hours is necessary for one conversion reaction. The main reasons are because the growth rate of the microorganisms is slow and because the enzyme reaction rate is slow since the VD3 is a nonnative substrate for the enzyme. It is thought that the active forms of VD3 of the same amount can be obtained in a shorter time by creating a variant enzyme with higher activity, and conducting the reaction in the microbial cell with a fast reproductive rate.(3)Presence of the product of side reaction: The greatest issue in the microbial conversion is a production of side reaction products where the carbon-26 is hydroxylated. This 26-hydroxyvitamin D3 (26(OH)VD3) is eluted as a proximal or overlapping peak of the 25(OH)VD3 in high-performance liquid chromatography. Therefore, to produce pharmaceutical quality 25(OH)VD3, it is necessary to remove the 26(OH)VD3 completely. This is a factor that reduces the yield of 25(OH)VD3. This is caused by the low regio-selectivity of the enzyme reaction, and it is necessary to create a variant enzyme with improved regio-selectivity to control the 26-hydroxylation.(4)Issue of cell membrane permeation: VD3 is a fat-soluble vitamin with poor water solubility. Therefore, as mentioned above, cyclodextrin (CD) is added to the conversion culture along with VD3, and VD3 is dissolved by trapping it in the cyclic structure of the CD. Since the CD-VD3 complex with relatively high molecular weight cannot permeate the cell membrane, it is necessary to devise a way where only the VD3 separated from CD can permeate the membrane, or is made to permeate along with the CD. The rate of cell membrane permeation is the rate-limiting factor of this microbial conversion, and if the issue of membrane permeation is improved, both Fig. 1 Production method for active form of vitamin D3Organic synthesis method (A) and microbial conversion method using P. autotrophica (B). HOHHHHOHHOHHOHHOHHOHBAAbout 20 steps(Drug)1α,25(OH)2VD31α,25(OH)2VD3CholesterolOHOH(Medical intermediate)25(OH)VD3(Drug)ExcretionP450(Vdh)P450(Vdh)OH(P. autotrophica)Pseudonocardia actinomycete ExcretionIntakeVitamin D3 (VD3)Inside cellOHOH

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