W. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 36–42
41
(863 Program) (2011AA02A211), the National Natural Sci-
ence Foundation of China (21276110,30970056), the Research
Fund for the Doctoral Program of Higher Education of China
(20110093120001), the Fundamental Research Funds for the Cen-
tral Universities (JUSRP51306A), the 111 Project (111-2-06) and a
Project Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions.
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Fig. 5. ADD accumulations with AD as substrate. Time course of AD transformation
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activity of KSDD expressed in B. subtilis could remain stable for a
long period. The further research suggested that the KSDD activity
mainly existed in intracellular, and it may ascribe to the trans-
membrane segments of this enzyme. It was previously reported
that the expressed KSDD in E. coli was mainly located in the inner
membrane in the form of inclusion bodies [16,17,22]. The over-
expressed KSDD from M. neoaurum JC-12 in E. coli with the vectors
pET28a and pET22b was executed in our original study, and the
recombinant proteins mainly existed in the form of inclusion bod-
ies. The enzyme activity of KSDD was not detected (on the vector
pET28a) or very weak (on the vector pET22b) in E. coli, and the
recombinant protein lost its activity in a very short time. From this
point of view, the B. subtilis system for KSDD expression is much
better than E. coli.
The percent conversion of AD by the recombinant B.
subtilis168/pMA5-ksdd was improved significantly, which was
about 18-fold higher than that of M. neoaurum JC-12. The percent
conversion of AD reached maximum in 10 h by the recombinant
B. subtilis 168, while it required 4–6 days by M. neoaurum. The
biosynthesis ADD in M. neoaurum contains the two steps: first, from
phytosterol to AD; then from AD to ADD. Compared to M. neoau-
rum, the B. subtilis 168 catalyzed the transformation of ADD from
AD in one-step. Moreover, the recombinant B. subtilis was cultured
much more easily than M. neoaurum. So the much higher conver-
sion of AD by the recombinant B. subtilis 168 was due to its rapid
growth and short steroids conversion periods. And most impor-
tantly, the B. subtilis was a very safe microorganism that is widely
used in industry.
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In conclusion, the enzyme KSDD from M. neoaurum JC-12 was
successfully expressed with stabilized enzyme activity in B. subtilis
168. The recombinant B. subtilis showed obviously superiority on
transformation of AD to ADD. Based on works above, the genes
related to ADD synthesis from phytosterol are considered to be
cloned in B. subtilis 168 to achieve one-step efficient transformation
of low-cost substrate to ADD, making the recombinant B. subtilis
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This work was supported by the Program for New Century
Excellent Talents in University (NCET-10-0459), the National
Basic Research Program of China (973 Program) (2012CB725202),
the High-tech Research and Development Programs of China