Microbial 11α-hydroxylation Of Androst-4-ene-3,17-dione And Microbial Transformation For Ginsenoside Rd Production

Microbial transformation has the advantages of high selectivity, low pollution and high efficiency. Hydroxylation is the most widespread type of steroid bioconversion which can be used to build intermediates for further chemical synthesis by offering access to otherwise inaccessible sites of the steroid molecule. Androst-4-ene-3,17-dione (AD) is an important intermediate of many steroid drugs such as corticosteroids, androgens, diuretics and anabolic agents. The oxygen group in position C11 is regarded as essential for anti-inflammatory action. The 11α-hydroxylation of androst-4-ene-3,17-dione leads to an intermediate in the production of Eplerenone (Trade name INSPRA), a selective aldosterone receptor blocking agent (SARA) which was developed by Pfizer and Pharmacia Company.Metarhizium anisopliae M28, which has the ability of introducing 11α-hydroxyl to its precursor androst-4-ene-3,17-dione (AD), was screened from various filamentous fungi. A mutant strain Metarhizium anisopliae M28-203 with high transformation efficiency was obtained by UV-LiCl mutation. The optimal fermentation culture determined by orthogonal test contains glucose 3%, corn steep 2%, silkworm chrysalis powder 0.5%, (NH4)2SO4 0.25%, K2HPO4 0.1%, MgSO4 0.05% and FeSO4 0.002%. The single factor experiments for temperature, pH, fermentation time, inoculum amount, substrate addition method and substrate concentration indicated that the optimal transformation condition was 28℃, pH6.0-6.5,5% inoculum amount,48-60 h mycelium age and 72 h transformation time. The conversion rate declined with the increase of substrate concentration because of the water-insolubility of steroid. Both 4% ethanol and 0.75 mg/mL Tween 80 were helpful for substrate solubility and then improve transformation efficiency. Conversion rate arrived above 65% under the optimal culture conditions when substrate concentration was 2 mg/mL (0.2%, w/v).Special problems such as low substrate concentration and transformation rate in steroid bioconversion originate from, in most cases, nearly total water-insolubility of the steroid substrates and difficulty to be transported inside the cell for biotransformation to take place. These can not be solved by organic reagents and surfactants because of their toxicity.β-cyclodextrin possesses a cage-like supramolecular structure with a hydrophobic cavity and hydrophilic outside and is able to form inclusion complexes with a wide variety of hydrophobic guest molecules and then improve their solubility. The effect ofβ-cyclodextrin on water-solubility of steroid substrate and hydroxylation by Metarhizium anisopliae M28-203 was investigated in this study. The results indicate that P-cyclodextrin could improve the solubility of androst-4-ene-3,17-dione in fermentation medium efficiently. Compared with 4% ethanol,8 g/L P-cyclodextrin could increase the conversion rate by 10% when the substrate concentration was 2 mg/mL(0.3%, w/v), furthermore the transformation time was shortened to 60 h. These suggest that the solubilization ofβ-cyclodextrin can facilitate the transportation of substrate inside the cell and then improve steroid bioconversion efficiency. Moreoverβ-cyclodextrin can improve substrate concentration to 3 mg/mL (0.3%, w/v) with the optimal molar ratio ofβ-cyclodextrin:AD of 1:1. Rotating frame overhauser effect spectroscopy (ROESY) was applied in structure determination ofβ-cyclodextrin:AD inclusion complex. The data indicate that the steroid skeletal is immersed in theβ-cyclodextrin cavity and stable inclusion complex is formed.Hydroxylase and hydroxylation mechanism of Metarhizium anisopliae M28-203 were investigated by protoplasts, cell-free extract and enzyme inhibitors. Hydroxylation by protoplasts of the strain indicated that its hydroxylase is endoenzyme which could be induced by steroid substrate and cell wall of this fungus is an important rate limitation factor. Both carbon monoxide difference spectrum of protoplasts, which revealed a maximum absorbance at 450 nm, and enzyme inhibition test of cell-free extract indicated that the steroid hydroxylase of Metarhizium anisopliae M28-203 belongs to cytochrome P450-dependent oxidoreductase system which includes cytochrome P450 and NADPH dependent cytochrome C reductase. The results on hydroxylation of androst-4-ene-3,17-dione (AD) by different subcellular fractions of the cell homogenate of Metarhizium anisopliae M28-203 suggested that two compositions of hydroxylase are distributed in different subcellular fractions and cytochrome P450 is presented in microsome. A high-yielding strain Metarhizium anisopliae M28-203 for lla-hydroxylation of androst-4-ene-3,17-dione (AD) was obtained, which provides a new biotransformation method for production of intermediate for Eplerenone and other steroid drugs. The inclusion complex of P-cyclodextrin and AD provides a benefit explore to improve substrate concentration. The investigation for hydroxylation mechanism of Metarhizium anisopliae M28-203 lays the groundwork on further purification of hydroxylase. Ginsenoside Rd is a rare protopanaxadiol ginsenoside which has various medicinal functions. Unfortunately, the content of ginsenoside Rd in wild ginseng is so low (less than 0.4%) that isolation of Rd from natural products is extremely difficult and costly. We have previously reported on a filamentous fungus Paecilomyces bainier 229 that effectively transforms ginsenoside Rbl to ginsenoside compound K (CK). A mutant Paecilomyces bainier 229-7 was obtained after UV-LiCl mutation and then selection on high concentration of ginsenosides substrate from the parent strain Paecilomyces bainier 229. Paecilomyces bainier 229-7 can transform ginsenoside Rbl to Rd with high selectivity and substrate tolerance and culture conditions required for biotransformation of ginsenoside Rbl to Rd under high substrate concentration had been optimized in 250 mL flask.In this study, Paecilomyces bainier 229-7 was applied in 10-L fermenter and the results further proved its high specificity and substrate tolerance. The mutant produces ginsenoside Rd from ginsenoside Rbl with as high as 88.9% bioconversion rate under optimized culture conditions in 10-L fermenter when supplied with 20 mg/mL of saponin from Panax notoginseng leaves (SPNL) by control of dissolved O2, agitation rate, pH and temperature。The isolation and purification of ginsenoside Rd from fermentation medium with macroporous resin was first reported. After macroporous resin HP20 crude separation, HP20 dehydration and H41 refine, the chromatographic purity and recovery of ginsenoside Rd arrived above 93% and 55%, respectively.Compared with other reported strains with high selectivity, Paecilomyces bainier 229-7 has the advantages of high substrate tolerance, high substrate concentration and short biotransformation time. Furthermore, the substrate used is SPNL extracted from stems and leaves of P. notoginseng (Burk.) F.H. Chen (Araliaceae), a Chinese herb often known by its common name, Tienchi ginseng, which is cheaper then pure ginsenoside Rbl. These results suggest that Paecilomyces bainier 229-7 could be useful for the preparation of ginsenoside Rd in the pharmaceutical industry.