14 16 constructed by codon optimization according to the cdna sequence of 3-ketosteroid 9α-hydroxylase of Rhodococcus erythropolis SQ1 reksha and Myco

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1 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov doi /j. issn α- 9α α- Mycobacterium tuberculosis H37Rv Rhodococcus erythropolis SQ α- reksha mtksha AD 9α α-OH-AD ReKshA MtKshA ReKshA α TB ReKshA BL21 DE3 -pet28a-reksha-yh μmol /L 0. 8% 0. 1 mmol /L IPTG 9α-OH-AD % α α- Q78 Q819 A Construction expression of 3-ketosteroid 9α-hydroxylase for 9α-OH-AD synthesis WANG Kang 1 2 WU Zhige 1 ZHAO Weirui 1 3 HU Sheng 1 MA Jumei 1 2 WANG Jinbo 1 YAO Shanjing 2 MEI Lehe School of Biotechnology and Chemical Engineering Ningbo Institute of Technology Zhejiang University Ningbo China 2. Department of Chemical and Biological Engineering Zhejiang University Hangzhou China 3. College of Biosystems Engineering and Food Science Zhejiang University Hangzhou China Abstract In order to develop a biocatalyst with high activity for 3 - ketosteroid 9α - hydroxylation recombinant strains BL21 DE3 - pet28a - ksha - reyh and BL21 DE3 - pet28a - ksha - mtyh were M GA LZ13B Q14C LY16B C A meilh@ zju.edu.cn.3- -9α J WANG Kang WU Zhige ZHAO Weirui et al.construction expression of 3-ketosteroid 9α-hydroxylase for 9α-OH-AD synthesis J.Chin J Bioprocess Eng

2 14 16 constructed by codon optimization according to the cdna sequence of 3-ketosteroid 9α-hydroxylase of Rhodococcus erythropolis SQ1 reksha and Mycobacterium tuberculosis H37Rv mtksha in the GenBank database. The soluble expression of ksha was significantly improved compared to unoptimized coding sequences. The recombinant strain was used to convert AD to 9α-OH-AD. The results showed that the ksha from Rhodococcus erythropolis SQ1 has significantly higher 9αhydroxylation activity towards anostenedione compared to that from Mycobacterium tuberculosis H37Rv. The yield reached % when BL21 DE3 - pet28a - ksha - reyh was used as catalyst under the conditions of 500 μmol /L AD 0. 1 mmol /L IPTG 0. 8% ethanol as cosolvent. The reaction was performed at 30 the substrate and IPTG were added at the same time. Keywords 4-androstene-3 17-dione AD 9α-hydroxy-4-androstene-3 17-dione 9α-OH-AD ksha hydroxylation steroid drug 9α α-OH-AD 9α-OH-AD 13 ksha 1-2 9α- 9α- 3-5 OH-AD 2 9α 9α α-Rhodococcus erythropolis SQ1 reksha Mycobacterium tuberculosis H37Rv mtksha KSH 6-9 KSH IA KshA KshB KshA RekshA KSH 9α AD 9α-OH-AD KshA 9α-OH-AD KSH 9α-OH-AD α- 9α-OH-AD 1. 1 ksha AD 9α-OH-AD 98% NADH 2006 pet-28a + Mycobacterium smegmatis mc2155 ksha E. coli DH5α Nhe Ⅰ Xho Ⅰ TaKaRa E. colibl DE3 T 4 DNA DNA BL21 Mycobacterium smegmatis 1. 5 μmol /L 1 Transgen 9α- PS 7 d mg /L 9α Rhodococcus rhodochrous 1. 2 KshA DSM ksha Jcat 14 http / /www. jcat. de ksha reksha mtksha reksha-yh mtksha-yh reksha mtksha Mycobaterrium neoaurum MwIB -01 ksha reksha-yh mtksha-yh

3 α ' 3' Nhe Ⅰ Xho Ⅰ 1. 3 Nhe I Xho I ksha pet28a + T 4 DNA mmol /L IPTG 200 μmol /L BL21 DE3 AD DMSO h BL21 DE3 -pet28a-mtksha BL21 DE3 -pet28a HPLC -reksha BL21 DE3 -pet28a - mtksha - yh BL21 DE3 -pet28a-reksha-yh 1. 4 KshA 200 μmol /L AD 0. 8% BL21 DE3 -pet28a-mtksha BL21 DE3 -pet28a -reksha BL21 DE3 -pet28a -mtksha -yh BL21 DE3 -pet28a -reksha -yh 4 2% 50 ml OD ~ mmol /L 50 μg /ml LB IPTG h OD ~ mmol /L IPTG r /min 6 h h PBS SDS -PAGE AD 9α-OH-AD BL21 DE3 -pet28a-reksha-yh BL21 DE3 -pet28a -mtksha-yh 2% TB 37 OD ~ 1. 0 IPTG 0. 5 mmol /L DMSO AD 300 μmol /L 30 Escherichia coli K % 24 h HPLC GUC Val CCC Pro reksha-yh 224 GC α-OH-AD HPLC r /min 2 min % HPLC 2. 2 KshA Hypersil ODS2 C mm 250 mm 5 μm V V = ml /min nm 20 μl pet28a-reksha BL21 DE3 -pet28a-mtksha-yh 1. 7 BL21 DE3 - pet28a - BL21 DE3 - pet28a - reksha - yh ksha-reyh 9α-OH-AD MtkshA RekshA LB TB OD ~ mmmol /L IPTG 300 μmol /L AD DMSO h 2 IPTG OD ~ OD ~ mmol /L IPTG DMSO h HPLC 500 μmol /L AD reksha mtksha mrna E. coli K12 GC reksha mtksha GC % 60. 7% GC % % mtkshayh 221 GC % 1 1 BL21 DE3 - pet28a - mtksha BL21 DE3-1 a 1 b reksha-

4 16 16 yh reksha 1 c 1 d mtksha - yh mtksha ' 2' 3' 4' Fig BL21 DE3 -pet28a-reksha BL21 DE3 -pet28a-reksha-yh BL21 DE3 -pet28amtksha BL21 DE3 -pet28a-mtksha-yh SDS-PAGE analysis of BL21 DE3 -pet28a-reksha BL21 DE3 -pet28a-reksha-yh BL21 DE3 -pet28a-mtksha and BL21 DE3 -pet28a-mtksha-yh 2. 3 AD 9α-OH-AD LC - IPTG 3 a MS 9α-OH-AD 3 a IPTG 0. 1 mmol /L 200 AD 2 μmol /L 24 h 9α-OH-AD % MtKshA AD 9α-OH-AD TB 24 h 9α-OH -AD 2. 7%ReKshA 3 b 3 b AD 9α - OH - AD % 200 μmol /L 24 h 9α-OH- MtKshA ReKshA AD % DMSO % AD 9α-OH-AD 2. 4 BL21 DE3 -pet28a-reksha-yh AD 9α-OH-AD AD 9α-OH-AD 4 4 AD DMSO 4 IPTG AD BL21 DE3 pet28a-reksha-yh LB 4 a OD α-OH-AD 2. 85% TB 9α-OH-AD 4 b OD α-OH-AD 85. 7% 9α-OH-AD

5 第6期 王 17 二酮 康等 3 甾酮 9α 羟基化酶基因的表达及其催化合成 9α 羟基雄甾 4 烯 3 图2 Fig. 2 AD 9α OH AD 的液质分析图谱 LC MS analysis of AD 9α OH AD and HPLC analysis of the relationship between peak area and the concentration of 9α OH AD 图3 Fig. 3 Effects of IPTG concentration and cosolvent on reaction yield 图4 Fig. 4 IPTG 浓度和助溶剂对反应得率的影响 底物添加时间对菌体生物量和反应得率的影响 Effects of time points of substrate addition on cell growth and productive yield 17

6 μmol /L 0. 8% 150 r /min IPTG h 9α-OH-AD % α Mycobacterium tuberculosis H37Rv Rhodococcus erythropolis SQ1 ksha AD Rhodococcus erythropolis SQ1 ksha BL21 DE3 -pet28a-ksha-reyh 9α BL21 DE3 -pet28a -ksha-reyh 9α -OH -AD 500 μmol /L 48 h 9α-OH -AD % 9α-OH -AD 9α-OH-AD ksha kshb ksha 1. 9α- Microbiol X P ANDOR A JEKKEL A HOPWOOD D A et al. Generation of useful insertionally blocked sterol degradation pathway mutants of fast - growing Mycobacteria and cloning characterization and expression of the terminal oxygenase of the 3 - ketosteroid 9α - hydroxylase in Mycobacterium smegmatis mc2155 J. Appl Environ Microbiol Nocardia canicruria BF313 9α- J HOLLAND H L. Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole- cell biocatalysts J.Steroids MAHATO S B GARAI S. Advances in microbial steroid biotransformation J.Steroids VANDER GEIZE R HESSELS G I VAN GERWEN R et al. Molecular and functional characterization of ksha and kshb encoding two components of 3 - ketosteroid 9α - hydroxylase a class IA monooxygenase in Rhodococcus erythropolis strain SQ1 J.Mol Microbiol ARNELL R JOHANNISSON R LINDHOLM J et al. Biotechnological approach to the synthesis of 9α - hydroxylated steroids J.Prep Biochem Biotechnol HU Y M VAN DER GEIZE R BESRA G S et al.3-ketosteroid 9α - hydroxylase is anessential factor in the pathogenesis of Mycobacterium tuberculosis J.Mol Microbiol STRIJEWSKI A. The steroid - 9α - hydroxylation system from Nocardia species J.Eur J Biochem DONOVA M V GULEVSKAYA S A DOVBNYA D V et al. Mycobacterium sp. mutant strain producing 9α - hydroxy and rostenedione from sitosterol J.Appl Genet Microbiol Biotechnol D PETRUSMA M DIJKHUIZEN L VAN DER GEIZE R. Rhodococcus rhodochrous DSM ketosteroid 9α - hydroxylase a two-component iron-sulfur-containing monooxygenase with subtle steroids substrate specificity J. Appl Environ 13.Mycobacterium neoaurum NwIB Δ α- D GROTE A HILLER K SCHEER M et al. JCat a novel tool to adapt codon usage of a target gene to its potential expression host J.Nucleic Acids Res W526-W531.