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[1]李梦 李雪梅 冯进辉** 张瑞 吴洽庆** 朱敦明 马延和.耻垢分枝杆菌中3-甾酮-Δ1-脱氢酶对植物甾醇转化积累9α羟基雄甾-4-烯-3,17-二酮的影响[J].应用与环境生物学报,2021,27(02):1-12.[doi:10.19675/j.cnki.1006-687x.2020.05037]
 LI Meng,LI Xuemei,FENG Jinhui**,et al.Effects of 3-ketosteroid-1-dehydrogenase on phytosterols transformation and the accumulation of 9-hydroxyandrost-4-ene-3,17-dione by Mycobacterium smegmatis[J].Chinese Journal of Applied & Environmental Biology,2021,27(02):1-12.[doi:10.19675/j.cnki.1006-687x.2020.05037]
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耻垢分枝杆菌中3-甾酮-Δ1-脱氢酶对植物甾醇转化积累9α羟基雄甾-4-烯-3,17-二酮的影响()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
27卷
期数:
2021年02期
页码:
1-12
栏目:
研究论文
出版日期:
2021-04-25

文章信息/Info

Title:
Effects of 3-ketosteroid-1-dehydrogenase on phytosterols transformation and the accumulation of 9-hydroxyandrost-4-ene-3,17-dione by Mycobacterium smegmatis
作者:
李梦12 李雪梅2 冯进辉2** 张瑞2 吴洽庆2** 朱敦明2 马延和2
1中国科学技术大学生命科学学院 合肥 230026
2中国科学院天津工业生物技术研究所 天津 300308
Author(s):
LI Meng12 LI Xuemei2 FENG Jinhui2** ZHANG Rui2 WU Qiaqing2** ZHU Dunming2 & MA Yanhe2
1School?of?Life?Sciences,University?of?Science?and?Technology?of?China,?Hefei?230026,?China. 2 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
关键词:
3-甾酮-Δ1-脱氢酶9α-羟基雄甾-4-烯-317-二酮植物甾醇生物转化
Keywords:
3-ketosteroid-1-dehydrogenase 9-hydroxyandrost-4-ene-317-dione phytosterols biotransformation
DOI:
10.19675/j.cnki.1006-687x.2020.05037
摘要:
9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)是一种重要的甾体药物中间体,分枝杆菌转化植物甾醇的过程中,9α-OH-AD的分解是导致其产率降低的一个关键因素,而3-甾酮-Δ1-脱氢酶(3-ketosteroid-Δ1-dehydrogenase,KstD)在9α-OH-AD的分解过程中起着重要的作用。耻垢分枝杆菌mc2155(Mycobacterium smegmatis mc 2155)菌株基因组中存在6个编码3-甾酮-Δ1-脱氢酶基因,结果显示单独失活基因kstD1,能够产生9α-OH-AD,但随着转化时间的延长9α-OH-AD会降解,无法积累。使用CRISPR-Cas12a辅助重组工程成功构建了同时失活kstD1、kstD2和kstD3的菌株,该菌株以5 g/L植物甾醇为底物时,转化14天内9α-OH-AD能够稳定存在,产物浓度达到2.86 g/L。本研究确定了耻垢分枝杆菌mc2155中对9α-OH-AD积累起着重要作用的3-甾酮-Δ1-脱氢酶,有效提高了菌种生产9α-OH-AD的稳定性和产率,可为构建9α-OH-AD生产菌种及产业化应用奠定基础。
Abstract:
9α-Hydroxyandrost-4-ene-3, 17-dione (9 αOH-AD) is an important steroid medicine intermediate . Decomposition of 9α-OH-AD is a key factor leading to low yield in the process of mycobacterium transforming phytosterols, and 3-ketosteroid-Δ1-dehydrogenase (KstD) may play an important role in the decomposition of 9 α-OH-AD. The objective is to identify the KstDs in Mycobacterium smegmatis mc2155 (M. smegmatis mc 2155) which are responsible for the decomposition of 9 α-OH-AD.There are six genes encoding 3-ketosteroid-Δ1-dehydrogenase in the genome of M. smegmatis mc 2155 strain, the results showed that the inactivation of kstD1 gene alone produce d 9 α-OH-AD, but 9α-OH-AD degraded and could not accumulate with the extension of the transformation time. The CRISPR-Cas12a assisted recombineering was used to successfully construct the strain that simultaneously inactivated kstD1, kstD2 and kstD3. When the strain was cultivated with phytosterols at 5 g/L substrate concentration , 9α-OH-AD can stably accumulate at 2.86 g/L concentration within 14 days. This study identified the 3-ketosteroid-Δ 1-dehydrogenase in M. smegmatis mc 2155 which are critical for the accumulation of 9α-OH-AD, and laid a foundation for the construction of strains for the effective production of 9 α-OH-AD.

相似文献/References:

[1]周龙飞,李会,许桠楠,等.油水双相体系中分枝杆菌降解植物甾醇产9-羟基雄烯二酮的工艺[J].应用与环境生物学报,2019,25(05):1200.[doi:10.19675/j.cnki.1006-687x.2019.01054]
 ZHOU Longfei,LI Hui**,XU Yanan,et al.Biotransformation of phytosterols to 9α-hydroxyandrostenedione by Mycobacterium sp. LY-1 in an oil-water two-phase system[J].Chinese Journal of Applied & Environmental Biology,2019,25(02):1200.[doi:10.19675/j.cnki.1006-687x.2019.01054]
[2]许桠楠,李会,周龙飞,等.Mycobacterium sp. LY-1高效转化植物甾醇两相体系的构建[J].应用与环境生物学报,2020,26(04):747.
 XU Yanan,LI Hui,ZHOU Longfei,et al.Constructing the two-phase system for the highly efficient transformation of phytosterols by Mycobacterium sp. LY-1[J].Chinese Journal of Applied & Environmental Biology,2020,26(02):747.
[3]李梦,李雪梅,冯进辉,等.耻垢分枝杆菌中3-甾酮-Δ1-脱氢酶对植物甾醇转化积累9α-羟基雄甾-4-烯-3,17-二酮的影响[J].应用与环境生物学报,2020,26(04):739.
 LI Meng,LI Xuemei,et al.Effects of the 3-ketosteroid-Δ1-dehydrogenase on phytosterol transformation with the accumulation of 9α-hydroxyandrost-4-ene-3,17-dione by Mycobacterium smegmatis[J].Chinese Journal of Applied & Environmental Biology,2020,26(02):739.

备注/Memo

备注/Memo:
收稿日期 Received: 2020-05-19 接受日期 Accepted: 2020-06-15
国家重点研发计划(2019YFA0905300)、天津市合成生物学技术创新能力提升行动(TSBICIP-KJGG-001-01,TSBICIP-KJGG-001-04)和中国科学院科技服务网络计划(KFJ-STS-QYZX-O66)资助
**通讯作者 Corresponding author (E-mail: wu_qq@tib.cas.cn; feng_jh@tib.cas.cn)
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更新日期/Last Update: 2020-07-03