| Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement | |
| Liu, Changshui1,3; Wang, Qi1,3; Xian, Mo1,2; Ding, Yamei4; Zhao, Guang1,2; Zhao, G | |
| 2013-09-20 | |
| 发表期刊 | PLOS ONE
 |
| ISSN | 1932-6203 |
| 卷号 | 8期号:9 |
| 文章类型 | Article |
| 摘要 | The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2) G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K-cat/K-m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.; The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2) G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K-cat/K-m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems. |
| 学科领域 | Science & Technology - Other Topics |
| DOI | 10.1371/journal.pone.0075554 |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Science & Technology - Other Topics |
| WOS类目 | Multidisciplinary Sciences |
| WOS记录号 | WOS:000324768000081 |
| WOS关键词 | RECOMBINANT ESCHERICHIA-COLI ; DEHYDROGENASE ; PROTEINS ; PATHWAY ; GENE ; BIOSYNTHESIS ; ACID |
| WOS标题词 | Science & Technology |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/16696 |
| 专题 | 海洋生物技术研发中心 |
| 通讯作者 | Zhao, G |
| 作者单位 | 1.Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao, Shandong, Peoples R China 2.Chinese Acad Sci, Key Lab Biobased Mat, Qingdao, Shandong, Peoples R China 3.Univ Chinese Acad Sci, Beijing, Peoples R China 4.Chinese Acad Sci, Inst Oceanol, Qingdao, Shandong, Peoples R China |
| 推荐引用方式 GB/T 7714 | Liu, Changshui,Wang, Qi,Xian, Mo,et al. Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement[J]. PLOS ONE,2013,8(9). |
| APA | Liu, Changshui,Wang, Qi,Xian, Mo,Ding, Yamei,Zhao, Guang,&Zhao, G.(2013).Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement.PLOS ONE,8(9). |
| MLA | Liu, Changshui,et al."Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement".PLOS ONE 8.9(2013). |
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| Dissection of Malony（926KB） | 限制开放 | CC BY-NC-SA | 浏览 | |||
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