Abstract: 2-Pyrrolidone as a highly promising bio-based platform chemical, has received more attention due to their widely used in textile and pharmaceutical industries. 2-Pyrrolidone synthesis pathway was established in Corynebacterium glutamicum by exploring the function of the key enzyme, CoA transferase, thus providing guidance for sustainable industrial synthesis of 2-pyrrolidone. Firstly, argB gene was knocked out to block the L-arginine biosynthesis pathway and increase the carbon flux to the 2-pyrrolidone synthesis pathway, resulting in a highly efficient synthesis of L-glutamate in the chassis cells. Secondly, a glutamate decarboxylase (Gad) was expressed to convert L-glutamate to GABA. Finally, CoA transferase derived from Anaerotignum propionicum was optimized for the N-terminus RBS, then recombined and expressed in C. glutamicum to convert GABA to 2-pyrrolidone. The engineered C.g EAGAN2 strain was tested in a 5 L fermenter and after 72 h of fermentation, (8±0.3) g/L 2-pyrrolidone was accumulated. The 2-pyrrolidone synthesis pathway was for the first time established in C. glutamicum. The CoA transferase could convert GABA to 2-pyrrolidone, achieving optimum biosynthesis in fed-batch cultures using glucose as a cheap carbon source.
 WERPY T A, HOLLADAY J E, WHITE J F. Top value added chemicals from biomass: I. results of screening for potential candidates from sugars and synthesis gas[R]. Synthetic Fuels, 2004,11(1): 926 125.  YAMANO N, TAKEDA S, NAKAYAMA A. Production of 2-pyrrolidone from biobased glutamate by using Escherichia coli[J]. Journal of Polymers and the Environment, 2013, 21(2):528-533.  PARK S J, KIM E Y, NOH W, et al. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli[J]. Bioprocess and Biosystems Engineering, 2013, 36(7):885-892.  TOKIWA Y, CALABIA B P, UGWU C U, et al. Biodegradability of plastics[J]. International Journal of Molecular Sciences, 2009, 10(9):3 722-3 742.  DABELSTEIN W, REGLITZKY A, ANDREA SCHUTZE, et al. Ullmann's encyclopedia of industrial chemistry[J]. Ullmanns Encyclopedia of Industrial Chemistry, 2016,09.DOI:10.1002/14356007.a16_719.pub3.  CHAE T U, KIM W J, CHOI S, et al. Metabolic engineering of Escherichia coli for the production of 1,3-diaminopropane, a three carbon diamine[J]. Scientific Reports, 2015, 5(1):13 040.  CHEN Y, NIELSEN J. Biobased organic acids production by metabolically engineered microorganisms[J]. Current Opinion in Biotechnology, 2016, 37:165-172.  ZHANG Jingwei, KAO E, KEASLING J D, et al. Metabolic engineering of Escherichia coli for the biosynthesis of 2-pyrrolidone[J]. Metabolic Engineering Communications, 2016, 3:1-7.  CHAE T U, KO Y S, HWANG K S, et al. Metabolic engineering of Escherichia coli for the production of four-, five- and six-carbon lactams[J]. Metabolic Engineering, 2017, 41:82-91.  XU Meijuan, RAO Zhiming, XU Zhenghong, et al. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production[J]. Amino Acids, 2012, 43(1):255-266.  CHOI J, YIM S, LEE S, et al. Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range[J]. Microbial Cell Factories, 2015, 14(1):21.  NGOC T H, TAEK J K. Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness[J]. Journal of Bioscience and Bioengineering, 2013, 115(2):154-158.  KITAOKA S, NAKANO Y. Colorimetric determination of omega-amino acids[J]. Journal of Biochemistry,1969, 66(1):87-94.  SONG C W, LEE J, KO Y S, et al. Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid[J]. Metabolic Engineering, 2015, 30(3):121-129.  刘艳,徐玉文,李玉梅.高效液相色谱法测定氨酪酸氯化钠注射液中的α-吡咯烷酮[J]. 中国生化药物杂志, 2012, 33(4):422-424.  SHI F, LUAN M, LI Y. Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum[J]. AMB Express, 2018, 8(1):61.  BARITUGO K G, KIM H T, DAVID T C, et al. Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery[J]. Biofuels, Bioproducts and Biorefining, 2018, 12(5):899-925.  BECKER J, ROHLES C M, WITTMANN C. Metabolically engineered Corynebacterium glutamicum for bio-based production of chemicals, fuels, materials, and healthcare products[J]. Metabolic Engineering, 2018, 50:122-141.  BECKER J, WITTMANN C. Bio-based production of chemicals, materials and fuels Corynebacterium glutamicum as versatile cell factory[J]. Current Opinion in Biotechnology, 2012, 23(4):631-640.  BARITUGO, KEI-ANNE, Kim, et al. Metabolic engineering of Corynebacterium glutamicum for fermentative production of chemicals in biorefinery[J]. Applied Microbiology and Biotechnology, 2018, 102(9):3 915-3 937.  WENDISCH V F, MINDT M, PÉREZ-GARCÍA, et al. Biotechnological production of mono- and diamines using bacteria: recent progress, applications, and perspectives[J]. Applied Microbiology and Biotechnology, 2018, 102(8):3 583-3 594.