Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions
文献类型：期刊论文
| 作者 | Liu, Xiutao1,2; Feng, Xinjun2; Ding, Yamei4; Gao, Wenjie2; Xian, Mo2; Wang, Jichao2; Zhao, Guang2,3 |
| 刊名 | METABOLIC ENGINEERING
 |
| 出版日期 | 2020-11-01 |
| 卷号 | 62页码:42-50 |
| ISSN号 | 1096-7176 |
| 关键词 | Propionyl-CoA carboxylase Carboxyl transferase Directed evolution Succinate biosynthesis CO2 fixation |
| DOI | 10.1016/j.ymben.2020.08.012 |
| 通讯作者 | Wang, Jichao(wangjc@qibebt.ac.cn) ; Zhao, Guang(zhaoguang@sdu.edu.cn) |
| 英文摘要 | Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccBBs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccBBs activity improvement, and a double mutant of PccBBs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by kcat/Km. Moreover, this PccBBs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO2 molecules, which was confirmed by isotope labeling experiment with NaH13CO3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO2 fixation reactions. |
| 资助项目 | NSFC[31722001] ; NSFC[31800081] ; NSFC[31961133014] ; CAS Key Program[ZDRW-ZS-2016-3M] ; Natural Science Foundation of Shandong Province[JQ201707] ; Shandong University |
| WOS研究方向 | Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| 出版者 | ACADEMIC PRESS INC ELSEVIER SCIENCE |
| WOS记录号 | WOS:000589825500005 |
| 源URL | [http://ir.qdio.ac.cn/handle/337002/169216]  |
| 专题 | 中国科学院海洋研究所 |
| 通讯作者 | Wang, Jichao; Zhao, Guang |
| 作者单位 | 1.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 2.Chinese Acad Sci, CAS Key Lab Biobased Mat, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao 266101, Peoples R China 3.Shandong Univ, State Key Lab Microbial Technol, Qingdao 266237, Peoples R China 4.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China |
| 推荐引用方式 GB/T 7714 | Liu, Xiutao,Feng, Xinjun,Ding, Yamei,et al. Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions[J]. METABOLIC ENGINEERING,2020,62:42-50. |
| APA | Liu, Xiutao.,Feng, Xinjun.,Ding, Yamei.,Gao, Wenjie.,Xian, Mo.,...&Zhao, Guang.(2020).Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions.METABOLIC ENGINEERING,62,42-50. |
| MLA | Liu, Xiutao,et al."Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions".METABOLIC ENGINEERING 62(2020):42-50. |
入库方式： OAI收割
来源：海洋研究所
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