Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions

doi:10.1016/j.ymben.2020.08.012

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	Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions
	Liu, Xiutao 1,2; Feng, Xinjun 1; Ding, Yamei 3; Gao, Wenjie 1; Xian, Mo 1; Wang, Jichao 1; Zhao, Guang 1,4
	2020-11-01
发表期刊	METABOLIC ENGINEERING
ISSN	1096-7176
卷号	62 页码:42-50
通讯作者	Wang, Jichao([email protected]) ; Zhao, Guang([email protected])
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.
关键词	Propionyl-CoA carboxylase Carboxyl transferase Directed evolution Succinate biosynthesis CO2 fixation
DOI	10.1016/j.ymben.2020.08.012
收录类别	SCI
语种	英语
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
WOS类目	Biotechnology & Applied Microbiology
WOS记录号	WOS:000589825500005
出版者	ACADEMIC PRESS INC ELSEVIER SCIENCE
引用统计	被引频次：18[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/169217
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.Chinese Acad Sci, CAS Key Lab Biobased Mat, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao 266101, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 3.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 4.Shandong Univ, State Key Lab Microbial Technol, Qingdao 266237, 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.

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