Vol.4 No.4 2012

39/62

Research paper : Efficient production of active form of vitamin D3 by microbial conversion (Y. Yasutake et al.)−229−Synthesiology - English edition Vol.4 No.4 (2012) the reaction efficiency (1) and the apparent reaction rate (2) can be expected to increase.Of the above issues that must be solved, issues (1) to (3) pertain to the performance of the enzyme that is actually involved in the conversion reaction. Therefore, characterization of the enzyme and improvement by introduction of the mutation, as well as the mass accumulation technology of the enzyme in the cell are necessary. As it will be explained later, this enzyme is one of the enzyme group called cytochrome P450. Cytochrome P450 is the name of the enzyme group that holds the heme in the molecule and has the ability to insert the hydroxyl group into the hydrocarbon chain of various substances by receiving external electrons. It requires appropriate electron-supplying protein to be active. Therefore, it is necessary to look for a redox partner gene that allows the efficient transfer of electrons to this enzyme, and this gene must be coexpressed along with the enzyme. Issue (4) is related to the structure of the cell membrane itself, or the function of the transporter protein that allows a substance to permeate the membrane, and the cell that can obtain such information is desired.To solve the above issues, we gathered data using the conversion host, which is a microorganism without the VD3 hydroxylation capacity, is capable of mass recombinant expression, is easy to culture, reproduces quickly, and whose genome information can be utilized. This information was fed back to the P. autotrophica system. As an organic species that fulfilled this condition, we decided to use the Rhodococcus erythropolis host-vector system[5] that belonged to the same actinomycetes as P. autotrophica (Fig. 2). 3 Road to results3.1 Isolation of the enzyme and identification of the geneIt was about 20 years ago when it was found that P. autotrophica, a rare actinomycete, possessed the ability to convert VD3 to 1,25(OH)2VD3. Due to the characteristic where it catalyzed the hydroxylation to the steroid skeleton, the enzyme that catalyzed this reaction was predicted to be a cytochrome P450, but the identification of the enzyme was not done successfully for a long time. Therefore, we started by searching the gene that encoded this enzyme. Since the genome sequence analysis had not been done for P. autotrophica, we attempted the purification of the enzyme directly from the cell extract using the VD3 hydroxylation activity as the index. In general, the VD3 hydroxylation activity could be detected by the coexistence of electron transferring protein that was needed for P450 to be active, and the target enzyme was confirmed as a P450 as predicted. However, purification was difficult due to the occurrence of the phenomenon where the VD3 hydroxylation activity became undetectable during the purification process. After much trial and error, we found that salt (NaCl and others) had to be present in the reaction solution for this enzyme to be active, and it was possible to follow the activity to the final step of purification[6]. After identifying the N-terminal and internal amino acid sequences from the purified enzyme, we succeeded in cloning the gene that encoded this enzyme. 3.2 Reproduction of the VD3 hydroxylation reaction in vitroIt was possible to produce the enzyme that showed VD3 hydroxylation activity, by using the general overexpression system of the E. coli. However, it was necessary to add 5-aminolevulinic acid, a heme precursor, to the culture media to obtain the heme-containing holo enzyme. This is a method commonly used for the mass production of P450 enzyme using E. coli as a host cell. On the other hand, this enzyme could be obtained using R. erythropolis without adding 5-aminolevulinic acid to the culture. This was thought to be because the actinomycetes possess many P450 genes, the heme biosynthesis pathway functioned stably, and the level of intracellular heme could be maintained without depletion. Since 5-aminolevulinic acid is an expensive reagent, the microbial conversion using R. erythropolis as a host cell is advantageous when conducting the recombinant expression.Next, the function of the enzyme was analyzed by in vitro reconstitution experiment. P450 required two electrons for one catalytic turnover of hydroxylation, and it was necessary to add the redox partner protein that supplied the electron to the assay system. Here, we used the commercially available redox partner proteins from spinach used widely for P450 assays. As a result, it was clarified that this enzyme continuously catalyzed the two-step hydroxylation from VD3 to 25(OH)VD3 and from 25(OH)VD3 to 1,25(OH)2VD3. Since 1(OH)VD3 was not detected, it was found that this Fig. 2 Outline of R&DIsolation and identification of enzyme and cloning of its encoding geneAchievement of high activity in enzymes, elimination of 26-hydroxylation side reactionEvolutionary engineering + 3D structureDevelopment of host-vector systemGenetic engineering technologiesImprovement of substrate permeability by modification of cell membraneSearch and improvement of optimal redox partner genesEngineering of P450 (Vdh)Construction of high efficiency vitamin D3 hydroxylation system by microbial cell

※このページを正しく表示するにはFlashPlayer9以上が必要です