Abstract: To clarify and compare the effects of enhancing the anaplerotic pathways on L-isoleucine production performance by Corynebacterium glutamicum. The genes of pyc and ppc were overexpressed by replacing the native promoter with Ptuf or by inserting the genes to plasmid in a L-isoleucine producer, C. glutamicum YI.The pyc overexpressed via genome-integration in ILE01 and via plasmid in ILE02 resulted in 17.3% (6.1 g/L) and 9.6% (5.7 g/L) increase in production of L-isoleucine by batch fermentation; as well as 11.7% (24.8 g/L) and 8.1% (24.0 g/L) by fed-batch fermentation. By comparison, the ppc overexpressed via the two strategies lead to 30.8% (6.8 g/L) and 13.5% (5.9 g/L), 15.8% (25.7 g/L) and 9.5% (24.3 g/L) increase by the two fermentation processes. Meanwhile, overexpression of pyc and ppc resulted in improvement of L-isoleucine yield. However, overexpression of pyc and ppc in ILE02 and ILE04 resulted in cell growth decreased. Overexpression of pyc and ppc both resulted in remarkably promotion of L-isoleucine production performance and the genome-integration overexpressing strategy was more profitable. This study first reports the effects of enhancing anaplerotic pathways on L-isoleucine production by C. glutamicum. The results would supply the reference for metabolic engineering of C. glutamicum.
PARK J H, OH J E, LEE K H, et al. Rational design of Escherichia coli for L-Isoleucine Production[J]. ACS Synthetic Biology, 2012, 1(11): 532-540.
LI Y, WEI H, WANG T, et al. Current status on metabolic engineering for the production of L-aspartate family amino acids and derivatives[J]. Bioresource Technology, 2017, 245: 1 588-1 602.
LEUCHTENBERGER W, HUTHMACHER K, DRAUZ K. Biotechnological production of amino acids and derivatives: Current status and prospects[J]. Applied Microbiology and Biotechnology, 2015, 69(1): 1-8.
CHEN Z, BOMMAREDDY R R, FRANK D, et al. Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum [J]. Applied and Environmental Microbiology, 2014, 80(4): 1 388-1 393.
GERSTMEIR R, WENDISCH VF, SCHNICKE S, et al. Acetate metabolism and its regulation in Corynebacterium glutamicum[J]. Journal of Biotechnology, 2003, 104(1-3): 99-122.
SAUER U, EIKMANNS B J. The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria[J]. FEMS Microbiology Reviews, 2005, 29(4): 765-794.
DELAUNAY S, UY D, BAUCHER M F, et al. Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation[J]. Metabolic Engineering, 1999, 1(4): 334-343.
SATO H, ORISHIMO K, SHIRAI T, et al. Distinct roles of two anaplerotic pathways in glutamate production induced by biotin limitation in Corynebacterium glutamicum[J]. Journal of Bioscience and Bioengineering, 2008, 106(1): 51-58.
GUO Xuan, WANG Jing, XIE Xixian, et al. Enhancing the supply of oxaloacetate for L-glutamate production by pyc overexpression in different Corynebacterium glutamicum[J]. Biotechnology Letters, 2013, 35(6): 943-950.
PETERS-WENDISCH P G, WENGISCH V F,DE GRAAF A A, et al. C3-carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum[J]. Archives of Microbiology, 1996, 165(6): 387-396.
GUBLER M, PARK S M, MIKE J, et al. Effects of phosphoenolpyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum[J]. Applied Microbiology and Biotechnology, 1994, 40(6): 857-863.
SAMBROOK J, MACCALLUM P, RUSSELL D. Molecular Cloning: A Laboratory Manual[M]. New York: Cold Spring Harbor Laboratory Press, 2001.
EGGELING L, BOTT M. Handbook of Corynebacterium glutamicum[M]. Boca Raton: CRC Press, 2005: 520-521.
BECKER J, ZELDER O, H & #xC3;FNER S, et al. From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production[J]. Metabolic Engineering, 2011, 13(2): 159-168.
LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25 (4): 402-408.
SAWADA K, ZEN-IN S, WADA M, et al. Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032[J]. Metabolic Engineering, 2010,12 (4): 401-407.
DE FORCHETTI S R M, CAZZULO J J. Some properties of the pyruvate carboxylase from Pseudomonas fluorescens[J]. Microbiology, 1976, 93 (1): 75-81.
YOKOTA A, SAWADA K, WADA M. Boosting anaplerotic reactions by pyruvate kinase gene deletion and phosphoenolpyruvate carboxylase desensitization for glutamic acid and lysine production in Corynebacterium glutamicum[J]. Advances in Biochemical Engineering-Biotechnology, 2017, 159: 181-198.
PETERS-WENDISCH P G, WENDISCH V F, PAUL S, et al. Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum[J]. Microbiology, 1997, 143(4): 1 095-1 103.
ZHANG Chenglin, LI Yuan, MA Jie, et al. High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistep metabolic engineering[J]. Metabolic Engineering, 2018, 49: 287-298.
PETERS-WENDISCH P, SCHIEL B, WENDISCH VF, et al. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum[J].Journal of Molecular Microbiology and Biotechnology, 2001, 3(2): 295-300.
KIND S, JEONG W K, SCHR & #xD5;DER H, et al. Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane[J]. Metabolic Engineering, 2010, 12(4): 341-351.
PETERSEN S, DE GRAAF A A, EGGELING L, et al. In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum[J]. Journal of Biological Chemistry, 2000, 275(46): 35 932-35 941.
NAGANO-SHOJI M, HAMAMOTO Y, MIZUNO Y, et al. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum[J]. Molecular Microbiology, 2017, 104(4): 677-689.
WADA M, SAWADA K, OGURA K, et al. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032[J].Journal of Bioscience and Bioengineering, 2016, 121(2): 172-177.