Overexpression of ribosome elongation factor G and recycling factor increases l-isoleucine production in Corynebacterium glutamicum
Tóm tắt
Ribosome elongation factor G encoded by fusA promotes the translocation step of protein synthesis in bacteria; ribosome recycling factor encoded by frr, together with the elongation factor G, dissociates ribosomes from messenger RNA after the termination of translation. Both factors play important roles during protein synthesis in bacteria. In this study, we found that overexpression of fusA and/or frr led to the increase of l-isoleucine production in Corynebacterium glutamicum IWJ001, an l-isoleucine production strain generated by random mutagenesis. Reverse transcription polymerase chain reaction analysis showed that transcriptional levels of genes lysC, hom, thrB, ilvA, ilvBN, and ilvE encoding the key enzymes in the biosynthetic pathway of l-isoleucine increased in C. glutamicum IWJ001 when fusA and/or frr were overexpressed. Co-overexpression of fusA and frr, together with genes ilvA, ilvB, ilvN, and ppnk in C. glutamicum IWJ001, led to 76.5 % increase of l-isoleucine production in flask cultivation and produced 28.5 g/L l-isoleucine in 72-h fed-batch fermentation. The results demonstrate that overexpressing ribosome elongation factor G and ribosome recycling factor is an efficient approach to enhance l-isoleucine production in C. glutamicum.
Tài liệu tham khảo
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254
Cremer J, Eggeling L, Sahm H (1991) Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl Environ Microbiol 57(6):1746–1752
Defeu S, Reimold C, Linne U, Knust T, Gescher J, Graumann PL (2010) Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proc Natl Acad Sci U S A 107(7):3163–3168
Eggeling I, Cordes C, Eggeling L, Sahm H (1987) Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to L-isoleucine. Appl Microbiol Biotechnol 25(4):346–351
Eggeling L, Morbach S, Sahm H (1997) The fruits of molecular physiology: engineering the L-isoleucine biosynthesis pathway in Corynebacterium glutamicum. J Biotechnol 56(3):167–182
Elisáková V, Pátek M, Holátko J, Nesvera J, Leyval D, Goergen JL, Delaunay S (2005) Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum. Appl Environ Microbiol 71(1):207–213
Guillouet S, Rodal AA, An GH, Lessard PA, Sinskey AJ (1999) Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production. Appl Environ Microbiol 65(7):3100–3107
Holtkamp W, Cunha C, Peske F, Konevega L, Wintermeyer W, Rodnina M (2014) GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits. EMBO J 33(9):1073–1085
Hosaka T, Xu J, Ochi K (2006) Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic-overproducing Streptomyces coelicolor ribosomal rpsL mutant. Mol Microbiol 61(4):883–897
Hou X, Chen X, Zhang Y, Qian H, Zhang W (2012) (L)-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum. Amino Acids 43(6):2301–2311
Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Krämer R, Linke B, McHardy AC, Meyer F, Möckel B, Pfefferle W, Pühler A, Rey DA, Rückert C, Rupp O, Sahm H, Wendisch VF, Wiegräbe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104(1–3):5–25
Katunin VI, Savelsbergh A, Rodnina MV, Wintermeyer W (2002) Coupling of GTP hydrolysis by elongation factor G to translocation and factor recycling on the ribosome. Biochemistry 41(42):12806–12812
Kavaliauskas D, Nissen P, Knudsen CR (2012) The busiest of all ribosomal assistants: elongation factor Tu. Biochemistry 51(13):2642–2651
Korös A, Varga Z, Molnár-Perl I (2008) Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A 1203(2):146–152
Lancaster L, Kiel MC, Kaji A, Noller HF (2002) Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing. Cell 111(1):129–140
Li L, Guo J, Wen Y, Chen Z, Song Y, Li J (2010) Overexpression of ribosome recycling factor causes increased production of avermectin in Streptomyces avermitilis strains. J Ind Microbiol Biotechnol 37(7):673–679
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) Method. Methods 25(4):402–408
Ma Z, Tao L, Bechthold A, Shentu X, Bian Y, Yu X (2014) Overexpression of ribosome recycling factor is responsible for improvement of nucleotide antibiotic-toyocamycin in Streptomyces diastatochromogenes 1628. Appl Microbiol Biotechnol 98(11):5051–5058
Maracci C, Peske F, Dannies E, Pohl C, Rodnina MV (2014) Ribosome-induced tuning of GTP hydrolysis by a translational GTPase. Proc Natl Acad Sci U S A 111(40):14418–14423
Miyajima R, Otsuka SI, Shiio I (1968) Regulation aspartate family amino acid biosynthesis in Brevibacterium flavum. J Biochem 63(2):139–148
Morbach S, Sahm H, Eggeling L (1995) Use of feedback-resistant threonine dehydratases of corynebacterium glutamicum to increase carbon flux towards l-isoleucine. Appl Environ Microbiol 61(12):4315–4320
Park JH, Lee SY (2010a) Metabolic pathways and fermentative production of L-aspartate family amino acids. Biotechnol J 5(6):560–577
Park JH, Lee SY (2010b) Fermentative production of branched chain amino acids: a focus on metabolic engineering. Appl Microbiol Biotechnol 85(3):491–506
Park JH, Oh JE, Lee KH, Kim JY, Lee SY (2012) Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol 1(11):532–540
Rolland N, Janosi L, Block MA, Shuda M, Teyssier E, Miège C, Chéniclet C, Carde JP, Kaji A, Joyard J (1999) Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in Escherichia coli carrying temperature-sensitive ribosome recycling factor. Proc Natl Acad Sci U S A 96:5464–5469
Savelsbergh A, Rodnina V, Wintermeyer W (2009) Distinct functions of elongation factor G in ribosome recycling and translocation. RNA 15(5):772–780
Schmeing TM, Ramakrishnan V (2009) What recent ribosome structures have revealed about the mechanism of translation. Nature 461(7268):1234–1242
Shi F, Li K, Huan X, Wang X (2013) Expression of NAD(H) kinase and glucose-6-phosphate dehydrogenase improve NADPH supply and L-isoleucine biosynthesis in Corynebacterium glutamicum ssp. lactofermentum. Appl Biochem Biotechnol 171(2):504–521
Shoji S, Walker SE, Fredrick K (2009) Ribosomal translocation: one step closer to the molecular mechanism. ACS Chem Biol 4(2):93–107
Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol 9(3):242–253
Teyssier E, Hirokawa G, Tretiakova A, Jameson B, Kaji A, Kaji H (2003) Temperature sensitive mutation in yeast mitochondrial ribosome recycling factor (RRF). Nucleic Acids Res 31:4218–4226
Vogt M, Krumbach K, Bang WG, van Ooyen J, Noack S, Klein B, Bott M, Eggeling L (2015) The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation. Appl Microbiol Biotechnol 99(2):791–800
Vrljic M, Kronemeyer W, Sahm H, Eggeling L (1995) Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants. J Bacteriol 177(14):4021–4027
Westerfeld WW (1945) A colorimetrie determination of blood acetoin. J Biol Chem 161:495–502
Xie X, Xu L, Shi J, Xu Q, Chen N (2012) Effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain Corynebacterium glutamicum YILW. J Ind Microbiol Biotechnol 39(10):1549–1556
Xu D, Tan Y, Huan X, Hu X, Wang X (2010) Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer. J Microbiol Methods 80(1):86–92
Xu L, Xie X, Shi J, Xu Q, Chen N (2013) Expression of the Escherichia coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw. Appl Biochem Microbiol 49(2):125–129
Yin L, Hu X, Xu D, Ning J, Chen J, Wang X (2012) Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum. Metab Eng 14(5):542–550
Yin L, Shi F, Hu X, Chen C, Wang X (2013) Increasing l-isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two-component export system BrnFE. J Appl Microbiol 114(5):1369–1377
Yin L, Hu X, Wang X (2014a) Proteomic analysis of L-isoleucine production by Corynebacterium glutamicum. J Pure Appl Microbiol 8(2):899–908
Yin L, Zhao J, Chen C, Hu X, Wang X (2014b) Enhancing the carbon flux and NADPH supply to increase L-isoleucine production in Corynebacterium glutamicum. Biotechnol Bioprocess Eng 19(1):132–142
Zhou J, Lancaster L, Donohue J, Noller H (2014) How the ribosome hands the A-site tRNA to the P site during EF-G-catalyzed translocation. Science 345(6201):1188–1191