Enhanced Steroid Metabolites Production by Resting Cell Phytosterol Bioconversion

Abstract

isms. 9 Hydroxypropyl-b-cyclodextrin (HP-b-CD), a CD derivative, has a greater capacity for complex formation than CD, and it has been recently used for the bioconversion of phytosterol to ADD and AD. 5,10 Recently, it was demonstrated that the bacterial strain Mycobacterium neoaurum NwIB-yV can be used in transforming phytosterol to 9-OH-AD. 11 The production of 9-OH-AD and the associated space-time yield reached 7.33 g L -1 and 1.22 g L -1 d -1 , respectively, from 15 g L -1 phytosterol in an aqueous system using growing M. neoaurum NwIB-yV cells, indicating a high potential for industrial appli-The steroid metabolites 9-hydroxy-androstenedione (9-OH-AD), androstadienedione (ADD) and androstenedione (AD) are important steroidal pharmaceuticals. In order to raise the production of steroid metabolites, an efficient resting cell phytosterol bioconversion process was developed to produce 9-OH-AD in the presence of hydroxypropyl-b-cyclodextrin (HP-b-CD). Cell growth medium containing phytosterol as an inducer positively improved cell activity. Under aerobic conditions, bioconversion proceeded at 70 g L -1 phytosterol in the presence of HP-b-CD (the optimized molar ratio of HP-b-CD/phytosterol was 1:1) with 30 g L -1 resting Mycobacterium neoaurum NwIB-yV cells (cell dry mass) in a 5-L bioreactor, where 9-OH-AD production and space-time yield reached 36.4 g L -1 and 9.1 g L -1 d -1 , respectively. The recycling of cells and HP-b-CD enables cost-saving and industrial applications. This bioprocess was also applied for the production of ADD and AD. The production of these steroid metabolites was much higher than that reported in previous studies.

Identification and in situ removal of an inhibitory intermediate to develop an efficient phytosterol bioconversion process using a cyclodextrin-resting cell system

“…In our previous study, Mycobacterium neoaurum NwIB-HK86 efficiently produced the main product 9α-OH-AD from phytosterols in HP-β-CD-resting cell system, accompanied by more than 20% of by-products such as androstenedione (AD), 9,22-dihydroxy-23,24-bisnorchol-4-en-3-one (DHBC), and 9,24-dihydroxychol-4-en-3-one (DHC). 20 Furthermore, the results demonstrated that the reaction was remarkably inhibited by high levels of substrates and metabolites. As shown in Fig.…”

Development of Engineered Ferredoxin Reductase Systems for the Efficient Hydroxylation of Steroidal Substrates

“…The 9OHAD production and space-time yield reached 19.64 g/L and 0.82 g/(L•h) from 20 g/L AD using whole cells of modi ed R. erythropolis RG1-UV29 strains without KstD activity [33]. Gao et al reported that the 9OHAD production and space-time yield could reach 36.4 g/L and 0.379 g/(L•h) from 70 g/L phytosterol with resting cells of M. neoaurum NwIB-yV in a 5-L bioreactor, respectively [12]. However, the production of 9OHAD could only reach 0.63 g/L (0.01 g/(L•h)) from 1 g/L AD using whole cells of recombinant E. coli [4], and 7.23 g/L (0.45 g/(L•h)) from 8 g/L AD using whole-cells recombinant B. subtilis, respectively [34].…”

“…45 Gao et al reported that the 9OHAD production and space−time yield could reach 36.4 g/ L and 0.379 g/(L•h) from 70 g/L phytosterol with 30 g/L resting cells of M. neoaurum NwIB-yV in a 5 L bioreactor. 46 However, 9OHAD production only reached 0.63 g/L [0.01 g/ (L•h)] from 1 g/L AD using growing cells of recombinant E. coli, 4 and 7.23 g/L [0.45 g/(L•h)] from 8 g/L AD using whole cells of recombinant B. subtilis. 47 Strain BLKA-RTM-F constructed in the present study was an excellent 9OHAD producer, with production of 5.24 g/L from 5 g/L AD using 50 g/L wet cells and space−time yield of 1.05 g/(L•h), a yield of 99.3% without byproducts.…”

“…Yao et al demonstrated a stable accumulation of 9-OHAD in a strain with a 7.33 g/L production from phytosterols by multiple deletions of three KstD genes and overexpression of the KshA gene in the M. neoaurum ATCC 25795 strain [11]. Gao et al reported that the 9OHAD production and space-time yield could reach 36.4 g/L and 9.1 g/(L⋅d) with resting cells of M. neoaurum NwIB-yV in a 5-L bioreactor, respectively [12].…”

Microbial Transformations of Plant Secondary Metabolites

“…Care has to be taken, particularly when growing cells are used, as high concentrations of surfactant may interfere with cell growth and activity [41]. The use of cyclodextrins and derivatives has also been reported as a tool to improve the solubility of hydrophobic substrates, particularly sterols [39,[42][43][44].…”