|本期目录/Table of Contents|

[1]杜好勉,诸葛斌,方慧英,等.利用甘油脱水酶基因构建产3-羟基丙醛工程菌及其表达比较[J].应用与环境生物学报,2013,19(01):20-24.[doi:10.3724/SP.J.1145.2013.00020]
 DU Haomian,ZHUGE Bin,FANG Huiying,et al.Construction of Recombinant Strains for 3-hydroxypropionaldehyde Biosynthesis and Comparison of Glycerol Dehydratase Gene Expression in Hosts*[J].Chinese Journal of Applied & Environmental Biology,2013,19(01):20-24.[doi:10.3724/SP.J.1145.2013.00020]
点击复制

利用甘油脱水酶基因构建产3-羟基丙醛工程菌及其表达比较()
分享到:

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
19卷
期数:
2013年01期
页码:
20-24
栏目:
研究论文
出版日期:
2013-02-25

文章信息/Info

Title:
Construction of Recombinant Strains for 3-hydroxypropionaldehyde Biosynthesis and Comparison of Glycerol Dehydratase Gene Expression in Hosts*
作者:
杜好勉诸葛斌方慧英张成宗红诸葛健
(1江南大学工业生物技术教育部重点实验室 无锡 214122)
(2江南大学食品科学与技术国家重点实验室 无锡 214122)
(3江南大学生物工程学院工业微生物研究中心 无锡 214122)
Author(s):
DU HaomianZHUGE BinFANG HuiyingZHANG ChengZONG HongZHUGE Jian
(1Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, Jiansu, China)
(2State Key Laboratory of Food Science and Technology of China, Jiangnan University, Wuxi 214122, Jiansu, China)
(3Research Center of Industrial Microorganisms, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiansu, China)
关键词:
甘油脱水酶3-羟基丙醛大肠杆菌tac启动子发酵
Keywords:
glycerol dehydratase 3-hydroxypropionaldehyde Escherichia coli tac promoter fermentation
分类号:
TQ920.1 : Q78
DOI:
10.3724/SP.J.1145.2013.00020
文献标志码:
A
摘要:
3-羟基丙醛(3-HPA)是一种重要的化工产品,可由甘油经甘油脱水酶作用后生成. 为获得产3-HPA基因工程菌,在已构建含甘油脱水酶基因及其激活因子大亚基质粒pEtac-dhaB-gdrA的基础上,构建了包含小亚基gdrB激活因子的重组质粒pEtac-dhaB-gdrA-gdrB. 利用大肠杆菌通用tac启动子将该质粒在不同Escherichia coli BL21、DH5a及JM109中进行表达. 阳性转化子经IPTG诱导后,提取总RNA,以cDNA为模板进行RT-PCR发现,目标基因在不同宿主都能较好转录. SDS-PAGE、酶活测定和3-HPA浓度测定结果表明,目标蛋白表达存在差异;酶活分别为4.7(±0.44)、3.5(±0.95)、8.1(±0.66) U/mg;发酵液中3-HPA的含量分别为0.012(±0.0044)、0.014(±0.003)、0.375(±0.018) g L-1,重组E. coli JM109/pEtac-dhaB-gdrA-gdrB具有较好的甘油脱水酶基因表达和产3-HPA性能. 该基因工程菌与克雷伯氏菌(Klebsiella pneumoniae)相比,发酵副产物明显较少,有利于后期提取,为生产3-HPA提供了一条新思路.
Abstract:
3-hydroxypropionaldehyde (3-HPA) is an important chemical product, which could be transformed from glycerol by glycerol dehydratase. In order to acquire an engineering strain to produce 3-HPA, gdrB encoding glycerol dehydratase reactivating factor small-subunit was amplified and employed to construct the plasmid pEtac-dhaB-gdrA-gdrB on the basis of the plasmid pEtac-dhaB-gdrA, which contained glycerol dehydratase reactivating factor large-subunit. The common Escherichia coli tac promoter was used for expression of pEtac-dhaB-gdrA-gdrB in different hosts E. coli BL21, DH5a, JM109. The positive clones were induced with IPTG, and the complete cDNA was obtained by RT-PCR using the total RNA as template. It was found that the gene of glycerol dehydratase could be transcribed well in all 3 hosts. The results of SDS-PAGE, enzyme analysis and the fermentation of 3-HPA indicated that the expression of glycerol dehydratase in different hosts were not the same. The specific enzyme activities of recombinant strains with different hosts (E. coli BL21, DH5a, JM109) were 4.7(±0.44), 3.5(±0.95) and 8.1(±0.66) U/mg, respectively, and the 3-HPA productions by shake flask fermentation were 0.012(±0.0044), 0.014(±0.003) and 0.375(±0.018) g L-1, respectively. The recombinant E. coli JM109/pEtac-dhaB-gdrA-gdrB had the best ability in glycerol dehydratase expression and 3-HPA production. Compared with Klebsiella pneumoniae, the by-products of recombinant E. coli were much less, which was good for separation and purification. This study provides a new route for the biosynthesis of 3-HPA.

参考文献/References:

1 Schaefer L, Auchtung T, Hermans K, Whitehead D, Borhan B, Britton R. The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups [J]. Microbiology, 2010, 156 (6): 1589-1599
2 张烽, 薛亚平, 郑裕国. 3-羟基丙醛的制备与应用[J]. 化学与生物工程, 2009, 26 (1): 58-60 [Zhang F, Xue YJ, Zheng YG. Preparation and application of 3-hydroxypropionaldehyde [J]. Chem Bioeng, 2009, 26 (1): 58-60]
3 Vancauwenberge JE, Slininger PJ, Bothast RJ. Bacterial conversion of glycerol to beta-hydroxypropionaldehyde [J]. Environ Microbiol, 1990, 56 (2): 329-332
4 Talarico TL, Casas IA, Chung TC, Dobrogose WJ. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri [J]. Antimicrob Agents Chemother, 1988, 32 (12): 1854-1858
5 权国燕, 方慧英, 诸葛斌,张波, 姚佳佳, 诸葛健. 甘油脱水酶再激活因子提高重组大肠杆菌3-羟基丙酸合成能力[J]. 中国生物工程杂志, 2011, 31 (6): 75-80 [Quan GY, Fang HY, Zhuge B, Zhang B, Yao JJ, Zhuge J. Glycerol dehydratase-reactivating factor increasing the recombinant Escherichia coli strains’ 3-hydroxypropinic acid synthesis capability [J]. China Biotechnol, 2011, 31 (6): 75-80]
6 梁宋平. 生物化学与分子生物学实验教程[M]. 北京: 高等教育出版社, 2003 [Liang SP. A Course of Biochemistry and Molecular Biology Experiment [M]. Beijing: Higher Education Press, 2003]
7 Ahrens K, Menzel K, Zeng AP, Deckwer WD. Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation [J]. Biotechnol Bioeng, 1998, 59 (5): 544-552
8 诸葛斌, 堵国成, 沈微, 诸葛健, 陈坚. 利用温控载体构建碱性果胶酯裂解酶工程菌[J]. 微生物学报, 2006, 46 (4): 657-659 [Zhuge B, Du GC, Shen W, Zhuge J, Chen J. Construction of an engineering strain producing alkaline pectate lyase with pHsh [J]. Acta Microbiol Sin, 2006, 46 (4): 657-659]
9 Slininger PJ, Bothast RJ, Smiley K. Production of 3-hydroxypropionaldehyde from glycerol [J]. Environ Microbiol, 1983, 46 (1): 62-67
10 Lüthi-Peng Q, Schärer S, Puhan Z. Production and stability of 3-hydroxypropionaldehyde in Lactobacillus reuteri [J]. Appl Microbiol Biotechnol, 2002, 60 (1): 73-80
11 陈宏文. 生物法合成1,3-丙二醇的过程工程研究[D]. 天津: 天津大学, 2005 [Chen HW. The research of 1,3-propanediol process engineering [D]. Tianjin: Tianjin University, 2005]
12 Makela J, Lloyd-Price J, Yli-Harja O, Ribeiro AS. Stochastic sequence-level model of coupled transcription and translation in prokaryotes [J]. BMC Bioinf, 2011, 12 (1): 121-133
13 Hargrove JL, Schmidt FH. The role of mRNA and protein stability in gene expression [J]. FASEB J, 1989, 3 (12): 2360-2370
14 罗杰. 细胞通透性的改变及其应用[J]. 微生物学报, 2001, 41 (3): 386-389 [Luo J. Cell permeabilized and its application [J]. Acta Microbiol Sin, 2001, 41 (3): 386-389]
15 Biebl H, Menzel K, Zeng AP, Deckwer WD. Microbial production of 1,3-propanediol [J]. Microbiol Biotechnol, 1999, 52 (3): 289-297
16 方慧英, 张成, 诸葛斌, 诸葛健. 产1,3-丙二醇新型基因工程菌的构建[J]. 应用与环境生物学报, 2009, 15 (5): 708-712 [Fang HY, Zhang C, Zhuge B, Zhuge J. Construction of novel recombinant strains capable of producing 1,3-propanediol [J]. Chin J Appl Environ Biol, 2009, 15 (5): 708-712]

相似文献/References:

[1]方慧英,张成,诸葛斌,等.产1,3-丙二醇新型基因工程菌的构建[J].应用与环境生物学报,2009,15(05):708.[doi:10.3724/SP.J.1145.2009.00708]
 FANG Huiying,ZHANG Cheng,ZHUGE Bin & ZHUGE Jian.Construction of Novel Recombinant Strains Capable of Producing 1,3-propanediol[J].Chinese Journal of Applied & Environmental Biology,2009,15(01):708.[doi:10.3724/SP.J.1145.2009.00708]
[2]聂玲燕,方慧英,诸葛斌,等.克雷伯氏菌产1,3-丙二醇菌株选育及甘油脱水酶基因转录分析[J].应用与环境生物学报,2013,19(01):25.[doi:10.3724/SP.J.1145.2013.00025]
 NIE Lingyan,FANG Huiying,ZHUGE Bing,et al.Breeding of Klebsiella for Production of 1,3-propanediol and Transcription Analysis of Glycerol Dehydratase Gene[J].Chinese Journal of Applied & Environmental Biology,2013,19(01):25.[doi:10.3724/SP.J.1145.2013.00025]
[3]马会亮,陈国,赵珺.罗伊氏乳杆菌中甘油脱水酶的催化特性及原位再激活[J].应用与环境生物学报,2013,19(01):30.[doi:10.3724/SP.J.1145.2013.00030]
 MA Huiliang,CHEN Guo,ZHAO Jun.Characteristics and in situ Reactivation of Glycerol Dehydratase in Lactobacillus reuteri[J].Chinese Journal of Applied & Environmental Biology,2013,19(01):30.[doi:10.3724/SP.J.1145.2013.00030]

备注/Memo

备注/Memo:
国家高技术研究发展计划(“863”计划,2006AA020103,2009AA02Z210)资助
更新日期/Last Update: 2013-02-26