重组大肠杆菌表达17β-羟基类固醇脱氢酶全细胞催化合成宝丹酮的研究

doi:10.11949/0438-1157.20200011

摘要/Abstract

摘要：

宝丹酮作为一种重要的蛋白同化雄性激素类固醇，具有提升肌肉质量和耐力的功能。宝丹酮的传统合成方法是以1,4-雄烯二酮（ADD）为底物通过化学法合成，但过程复杂、污染严重。17β-羟基类固醇脱氢酶（17β-HSD）可催化甾体化合物C-17位点的氧化还原反应，实现ADD和宝丹酮的相互转化。本研究通过基因序列同源性分析，筛选到6种不同来源的17β-HSD基因并对其在大肠杆菌中进行异源表达。利用不同重组菌转化ADD合成宝丹酮，结果表明重组菌BL21/pET28a-HSDPy的ADD转化率最高，因此选择BL21/pET28a-HSDPy进行进一步研究。鉴定了重组菌的酶学性质并优化其全细胞转化条件。结果表明在生物量为36 g·L^-1、底物浓度为5.40 g·L^-1条件下，经过两次补料，获得了3.66 g·L^-1宝丹酮，比优化前提高了4.1倍。而且在生物转化过程中未检测到副产物。为生物合成宝丹酮提供了可能。

关键词: 17β-羟基类固醇脱氢酶, 1,4-雄烯二酮, 宝丹酮, 生物催化

Abstract:

Boldenone, as an important anabolic androgenic steroid, has the function of improving muscle mass and endurance. The traditional synthesis method of boldenone is to synthesize from 1,4-androstenedione (ADD) by chemical method, but the process is complex and the pollution is serious. 17β-hydroxysteroid dehydrogenase (17β-HSD) can catalyze the redox reaction of C-17 site of steroids, and realize the conversion of ADD and boldenone. In this study, six different 17β-HSD genes were screened and heterologously expressed in E. coli. Using different recombinant strains to transform ADD to boldenone, the results showed that E. coli BL21/pET28a-HSDPy had the highest conversion rate of ADD, so BL21/pET28a-HSDPy was selected for further study. The enzymatic properties of the recombinant strain were identified and the whole cell transformation conditions were optimized. The results showed that under the condition of 36 g·L^-1 biomass and 5.40 g·L^-1 substrate concentration, 3.66 g·L^-1 boldenone was obtained after two batches of feeding, which was 4.1 times higher than before optimization. Moreover, no by-products were detected during biotransformation. This study provides the possibility for the biosynthesis of boldenone.

Key words: 17β-hydroxysteroid dehydrogenase, 1,4-androstenedione, boldenone, biocatalysis

中图分类号:

Q 814.9

吴玉玲, 邵明龙, 周武林, 高惠芳, 张显, 徐美娟, 杨套伟, 饶志明. 重组大肠杆菌表达17β-羟基类固醇脱氢酶全细胞催化合成宝丹酮的研究[J]. 化工学报, 2020, 71(7): 3229-3237.

Yuling WU, Minglong SHAO, Wulin ZHOU, Huifang GAO, Xian ZHANG, Meijuan XU, Taowei YANG, Zhiming RAO. Study on catalytic synthesis of boldenone by recombinant E. coli expressing 17β-hydroxysteroid dehydrogenase[J]. CIESC Journal, 2020, 71(7): 3229-3237.

图/表 10

图1 17β-羟基类固醇脱氢酶（17β-HSD）生物催化ADD合成BD

Fig.1 Bioconversion of ADD to BD by 17β-hydroxysteroid dehydrogenase (17β-HSD)

表1 本研究所用到的菌株、质粒及引物

Table 1 Strains, plasmids and primers used in this study

菌株、质粒或引物	性质	来源
菌株
Escherichia coli
JM109	用于所有克隆实验	Invitrogen
BL21 (DE3)	重组蛋白表达宿主	Promega
BL21/pET28a-HSDCl	重组E. coli BL21 (DE3) ，携带pET28a- HSDCl	本研究构建
BL21/pET28a-HSDPy	重组E. coli BL21 (DE3) ，携带pET28a- HSDPy	本研究构建
BL21/pET28a-HSDBb	重组E. coli BL21 (DE3) ，携带 pET28a- HSDBb	本研究构建
BL21/pET28a-HSDAo	重组E. coli BL21 (DE3) ，携带 pET28a- HSDAo	本研究构建
BL21/pET28a-HSDCt	重组E. coli BL21 (DE3) ，携带pET28a- HSDCt	本研究构建
BL21/pET28a-HSDBu	重组E. coli BL21 (DE3) ，携带pET28a- HSDBu	本研究构建
质粒
pUC57- HSDCl	pUC57携带来源于Cochliobolus lunatus经过密码子优化的17β-HSD基因，Amp^R	金唯智合成
pUC57- HSDPy	pUC57携带来源于Pyrenochaeta sp.经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pUC57- HSDBb	pUC57携带来源于Beauveria bassiana经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pUC57- HSDAo	pUC57携带来源于Arthroderma otae经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pUC57- HSDCt	pUC57携带来源于Comamonas testosterone经过密码子优化的17β-HSD基因，Amp^R	金唯智合成
pUC57- HSDBu	pUC57携带来源于Burkholderia sp.经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pET28a(+)	大肠杆菌表达载体，Kan^R	Novagen
pET28a-HSDCl	pET28a携带来源于Cochliobolus lunatus的17β-HSD基因， Kan^R	本研究构建
pET28a-HSDPy	pET28a携带来源于Pyrenochaeta sp.的17β-HSD基因， Kan^R	本研究构建
pET28a-HSDBb	pET28a携带来源于Beauveria bassiana的17β-HSD基因，Kan^R	本研究构建
pET28a-HSDAo	pET28a携带来源于Arthroderma otae的17β-HSD基因，Kan^R	本研究构建
pET28a-HSDCt	pET28a携带来源于Comamonas testosterone的17β-HSD基因，Kan^R	本研究构建
pET28a-HSDBu	pET28a携带来源于Burkholderia sp.的17β-HSD基因，Kan^R	本研究构建
引物	序列（5′-3′）
HSDCl-F	CGGGATCCATGCCGCACGTGGAGAACGCC (BamH I)
HSDCl-R	CCAAGCTTTCACGCCGCGCCACCGTCTAAAG (Hind III)
HSDPy-F	CGGGATCCATGTCGGACCAGTATGTCCCGAACCGTTTAGAGG (BamH I)
HSDPy-R	CCAAGCTTT CACGCCGCGCCGCCGTCTAAAG (Hind III)
HSDBb-F	CGGGATCCATGACGGGCGCCACCACCA (BamH I)
HSDBb-R	CCAAGCTTTCAGGCGGCGCCGCCGTC (Hind III)
HSDAo-F	CGGGATCCATGGGCTCGGTCTCGTATATCCCG (BamH I)
HSDAo-R	CCAAGCTTTCAGGCGGCGCCGCCGTC (Hind III)
HSDCt-F	CGGGATCCATGACCAACCGGCTGCAAGGTAAGG (BamH I)
HSDCt-R	CCAAGCTTTCATAAACCCATGCCTAAAATCGAGTTGTCGG (Hind III)
HSDBu-F	CGGGATCCATGGGCGACCGGCTGAAG (BamH I)
HSDBu-R	CCAAGCTTTCACGCGATGCCCATGATC (Hind III)

表1 本研究所用到的菌株、质粒及引物

Table 1 Strains, plasmids and primers used in this study

菌株、质粒或引物	性质	来源
菌株
Escherichia coli
JM109	用于所有克隆实验	Invitrogen
BL21 (DE3)	重组蛋白表达宿主	Promega
BL21/pET28a-HSDCl	重组E. coli BL21 (DE3) ，携带pET28a- HSDCl	本研究构建
BL21/pET28a-HSDPy	重组E. coli BL21 (DE3) ，携带pET28a- HSDPy	本研究构建
BL21/pET28a-HSDBb	重组E. coli BL21 (DE3) ，携带 pET28a- HSDBb	本研究构建
BL21/pET28a-HSDAo	重组E. coli BL21 (DE3) ，携带 pET28a- HSDAo	本研究构建
BL21/pET28a-HSDCt	重组E. coli BL21 (DE3) ，携带pET28a- HSDCt	本研究构建
BL21/pET28a-HSDBu	重组E. coli BL21 (DE3) ，携带pET28a- HSDBu	本研究构建
质粒
pUC57- HSDCl	pUC57携带来源于Cochliobolus lunatus经过密码子优化的17β-HSD基因，Amp^R	金唯智合成
pUC57- HSDPy	pUC57携带来源于Pyrenochaeta sp.经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pUC57- HSDBb	pUC57携带来源于Beauveria bassiana经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pUC57- HSDAo	pUC57携带来源于Arthroderma otae经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pUC57- HSDCt	pUC57携带来源于Comamonas testosterone经过密码子优化的17β-HSD基因，Amp^R	金唯智合成
pUC57- HSDBu	pUC57携带来源于Burkholderia sp.经过密码子优化的17β-HSD基因， Amp^R	金唯智合成
pET28a(+)	大肠杆菌表达载体，Kan^R	Novagen
pET28a-HSDCl	pET28a携带来源于Cochliobolus lunatus的17β-HSD基因， Kan^R	本研究构建
pET28a-HSDPy	pET28a携带来源于Pyrenochaeta sp.的17β-HSD基因， Kan^R	本研究构建
pET28a-HSDBb	pET28a携带来源于Beauveria bassiana的17β-HSD基因，Kan^R	本研究构建
pET28a-HSDAo	pET28a携带来源于Arthroderma otae的17β-HSD基因，Kan^R	本研究构建
pET28a-HSDCt	pET28a携带来源于Comamonas testosterone的17β-HSD基因，Kan^R	本研究构建
pET28a-HSDBu	pET28a携带来源于Burkholderia sp.的17β-HSD基因，Kan^R	本研究构建
引物	序列（5′-3′）
HSDCl-F	CGGGATCCATGCCGCACGTGGAGAACGCC (BamH I)
HSDCl-R	CCAAGCTTTCACGCCGCGCCACCGTCTAAAG (Hind III)
HSDPy-F	CGGGATCCATGTCGGACCAGTATGTCCCGAACCGTTTAGAGG (BamH I)
HSDPy-R	CCAAGCTTT CACGCCGCGCCGCCGTCTAAAG (Hind III)
HSDBb-F	CGGGATCCATGACGGGCGCCACCACCA (BamH I)
HSDBb-R	CCAAGCTTTCAGGCGGCGCCGCCGTC (Hind III)
HSDAo-F	CGGGATCCATGGGCTCGGTCTCGTATATCCCG (BamH I)
HSDAo-R	CCAAGCTTTCAGGCGGCGCCGCCGTC (Hind III)
HSDCt-F	CGGGATCCATGACCAACCGGCTGCAAGGTAAGG (BamH I)
HSDCt-R	CCAAGCTTTCATAAACCCATGCCTAAAATCGAGTTGTCGG (Hind III)
HSDBu-F	CGGGATCCATGGGCGACCGGCTGAAG (BamH I)
HSDBu-R	CCAAGCTTTCACGCGATGCCCATGATC (Hind III)

图2 17β-HSD的同源性分析和序列比对

Fig.2 Homology analysis and sequence alignment of 17β-HSD

图3 重组质粒pET28a-HSD酶切验证M—DNA Marker；1,3,5,7,9,11—pET28a-HSDCl，pET28a-HSDPy，pET28a-HSDBb，pET28a-HSDAo，pET28a-HSDCt，pET28a-HSDBu通过BamH I酶切；2,4,6,8,10,12—pET28a-HSDCl，pET28a-HSDPy，pET28a-HSDBb，pET28a-HSDAo，pET28a-HSDCt，pET28a-HSDBu通过BamHI和 Hind III酶切

Fig.3 Identification of recombinant pET28a-HSD by enzyme digestion

图4 重组大肠杆菌17β-HSD SDS-PAGE分析M—蛋白Marker；1—重组大肠杆菌BL21/pET28a粗酶液；2—重组大肠杆菌BL21/pET28a-HSDCl粗酶液；3—重组大肠杆菌BL21/pET28a-HSDPy粗酶液；4—重组大肠杆菌BL21/pET28a-HSDBb粗酶液；5—重组大肠杆菌BL21/pET28a-HSDAo粗酶液；6—重组大肠杆菌BL21/pET28a-HSDCt粗酶液；7—重组大肠杆菌BL21/pET28a-HSDBu粗酶液

Fig.4 SDS-PAGE analysis of 17β-HSD expression in recombinant E. coli

表2 重组大肠杆菌BL21/pET28a-HSD转化ADD

Table 2 Conversion of ADD by recombinant E. coli BL21/pET28a-HSD

菌株	12 h转化率/%	来源
BL21/pET28a	—	—
BL21/pET28a-HSDCl	54.90 $± 2.15$	Cochliobolus lunatus
BL21/pET28a-HSDPy	72.53 $± 1.36$	Pyrenochaeta sp.
BL21/pET28a-HSDBb	23.12 $± 2.89$	Beauveria bassiana
BL21/pET28a-HSDAo	10.11 $± 1.08$	Arthroderma otae
BL21/pET28a-HSDCt	9.39 $± 1.45$	Comamonas testosterone
BL21/pET28a-HSDBu	9.03 $±$ 0.72	Burkholderia sp.

表2 重组大肠杆菌BL21/pET28a-HSD转化ADD

Table 2 Conversion of ADD by recombinant E. coli BL21/pET28a-HSD

菌株	12 h转化率/%	来源
BL21/pET28a	—	—
BL21/pET28a-HSDCl	54.90 $± 2.15$	Cochliobolus lunatus
BL21/pET28a-HSDPy	72.53 $± 1.36$	Pyrenochaeta sp.
BL21/pET28a-HSDBb	23.12 $± 2.89$	Beauveria bassiana
BL21/pET28a-HSDAo	10.11 $± 1.08$	Arthroderma otae
BL21/pET28a-HSDCt	9.39 $± 1.45$	Comamonas testosterone
BL21/pET28a-HSDBu	9.03 $±$ 0.72	Burkholderia sp.

图6 不同金属离子及EDTA对HSDPy酶活性的影响

Fig.6 Effect of different metal ions and EDTA on activity of HSDPy

图7 大肠杆菌BL21/pET28a-HSDPy的不同生物量（a）和不同底物浓度（b）对宝丹酮生产的影响

Fig.7 Effects of different biomasses of E. coli BL21/pET28a-HSDPy (a) and substrate concentration (b) on BD production

图5 pH（a）和温度（b）对HSDPy酶活性的影响

Fig.5 Effects of pH (a) and temperature (b) on activity of HSDPy

图8 分批转化（a）和补料分批转化（b）ADD合成宝丹酮的过程

Fig.8 Batch conversion (a) and fed-batch conversion (b) of ADD to synthesize BD

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