title: Effect of Polyhydroxybutyrate (PHB) storage on L-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation.
abstract: BACKGROUND
  Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5.
  
  
  RESULTS
  C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain.
  
  
  CONCLUSIONS
  Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield L-arginine.
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. xumeijuan@jiangnan.edu.cn.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. 94046845@qq.com.
authors.forename: Jingru
authors.initials: J
authors.lastname: Qin
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. raozhm@jiangnan.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. hengyi.you@gmail.com.
authors.forename: Hengyi
authors.initials: H
authors.lastname: You
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. zxshengwu@126.com.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. ytw1228@163.com.
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. xiaoyuanwang@hotmail.com.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. zhenghxu@163.com.
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
mesh_terms: D001120:Arginine / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D003352:Corynebacterium / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006885:Hydroxybutyrates / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D001120:Arginine; D001426:Bacterial Proteins; D003067:Coenzymes; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates