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Research paper−227−Synthesiology - English edition Vol.4 No.4 pp.227-235 (Mar. 2012) method, it is difficult to conduct regio-selective hydroxylation for the steroid skeleton, and it is not a suitable method for manufacturing active form of VD3 and its derivatives that may be pharmacologically effective.As an alternative to this chemical synthesis method, the manufacture of 1,25(OH)2VD3 using the microbial conversion capability has been realized[3][4]. The actinomycete Pseudonocardia autotrophica that conducts this microbial conversion has the capacity to convert the VD3 added to the culture into 1,25(OH)2VD3. Moreover, the reaction intermediate 25(OH)VD3 produced in this microbial conversion process is a valuable medical intermediate with the pharmacological effect equivalent to 1,25(OH)2VD3, and the 25(OH)VD3 can be obtained in the same process. The price of 25(OH)VD3 sold as general-use reagent is about 40,000 yen per 1 mg, according to the catalog of Company S (Fig. 1B).The biocatalytic conversion using the enzymes of organisms, in general, shows high regio-selectivity and stereoselectivity, and has major impact on the synthesis of chemical substances. Also, the biocatalytic conversion is a safe method compared to the conventional organic synthesis, in that it has very low emission of pollutants under mild reaction condition (ordinary temperature and pressure), and involves low energy consumption. The term “green chemistry” is used for the biocatalytic conversion technology with the aforementioned characteristics, as it is known as an environment-friendly synthesis. The production of active forms of VD3 by P. autotrophica is an environment-friendly production method with all of 1 IntroductionVitamin D3 (VD3) is a fat-soluble secosteroid hormone involved in various important physiological functions in the human body[1]. Humans take in most of the VD3 from food, and the ingested VD3 is converted into active forms of VD3 (25-hydroxyvitamin D3 (25(OH)VD3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2VD3)) in the liver and kidneys. These active forms of VD3 are deeply involved in maintenance of calcium and phosphate homeostasis, cell reproduction and differentiation, immunity adjustment, and other functions in the human body. The deficiency of active forms of VD3 due to genetic or environmental factors is known to cause diseases such as osteoporosis, rachitis, psoriasis, and hyperparathyroidism, and in fact, active forms of VD3 are used as the treatment drug for such diseases[1].Currently, the 1,25(OH)2VD3 mainly used as drugs is manufactured by a chemical synthesis method, and it can be synthesized in approximately 20 reaction steps with cholesterol as the starting substance (Fig. 1A). However, the yield is only about 1 %[2]. It is commercially manufactured despite such low production efficiency, since the active form of VD3 shows pharmacological effect at very low dose (0.5~several g/day or less), but the price is extremely high. While the price of VD3 sold as general-use reagent is about 7 yen per 1 mg, the price of 1,25(OH)2VD3 is about 130,000 yen/mg, according to the catalog of Company S. To mass-produce such fine chemicals, the manufacture method becomes complex due to low reaction efficiency in the aforementioned chemical synthesis, and high cost must be paid to manufacture highly pure product that can be used as pharmaceuticals. Also, in the chemical synthesis - Comprehensive approach from the molecular to the cellular level-Conversion processes of organic compounds using biocatalyst generally have high regio- and stereo-selectivity, and are becoming increasingly important for efficient production of chemicals. In addition, biocatalysis is less hazardous, less polluting and less energy-consuming than the conventional chemical method. We report the highly efficient bioconversion system using actinomycete Rhodococcus erythropolis to produce active form of vitamin D3 currently used as a pharmaceutical. The improvement of performance of the enzyme used for the bioconversion has been achieved by the combination of evolutionary engineering and structure-based methods. Accordingly, the practical production efficiency of active form of vitamin D3 has been substantially increased. In addition, we have succeeded in significant improvement of cellular permeability of vitamin D3 by using nisin-treated cells, and have developed a new platform for vitamin D3 hydroxylation process. Efficient production of active form of vitamin D3 by microbial conversion Keywords : Cytochrome P450, vitamin D3, bioconversion, nisin, structural biology, protein engineering [Translation from Synthesiology, Vol.4, No.4, p.222-229 (2011)]Yoshiaki Yasutake and Tomohiro Tamura*Bioproduction Research Institute, AIST 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan *E-mail : Original manuscript received September 7, 2011, Revisions received October 31, 2011, Accepted November 7, 2011

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