Alleviation of carbon catabolite repression in Enterobacter aerogenes for efficient utilization of sugarcane molasses for 2,3-butanediol production

Biotechnology for Biofuels

Moo-Young Jung²  Hwi-Min Jung²  Jinwon Lee¹  Min-Kyu Oh² 

[1] Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Republic of Korea

[2] Department of Chemical and Biological Engineering, Korea University, Seoul, Republic of Korea

关键词: Carbon catabolite repression;    Catabolite repressor/activator;    Fed-batch fermentation;    Sugarcane molasses;    Enterobacter aerogenes;    2,3-Butanediol;   

Others  :  1225781
DOI  :  10.1186/s13068-015-0290-3

 received in 2015-02-17, accepted in 2015-07-22,  发布年份 2015

【 摘 要 】

Background

Due to its cost-effectiveness and rich sugar composition, sugarcane molasses is considered to be a promising carbon source for biorefinery. However, the sugar mixture in sugarcane molasses is not consumed as efficiently as glucose in microbial fermentation due to complex interactions among their utilizing pathways, such as carbon catabolite repression (CCR). In this study, 2,3-butanediol-producing Enterobacter aerogenes was engineered to alleviate CCR and improve sugar utilization by modulating its carbon preference.

Results

The gene encoding catabolite repressor/activator (Cra) was deleted in the genome of E. aerogenes to increase the fructose consumption rate. However, the deletion mutation repressed sucrose utilization, resulting in the accumulation of sucrose in the fermentation medium. Cra regulation on expression of the scrAB operon involved in sucrose catabolism was verified by reverse transcription and real-time PCR, and the efficiency of sucrose utilization was restored by disrupting the scrR gene and overexpressing the scrAB operon. In addition, overexpression of the ptsG gene involved in glucose utilization enhanced the glucose preference among mixed sugars, which relieved glucose accumulation in fed-batch fermentation. In fed-batch fermentation using sugarcane molasses, the maximum titer of 2,3-butanediol production by the mutant reached 140.0 g/L at 54 h, which was by far the highest titer of 2,3-butanediol with E. aerogenes achieved through genetic engineering.

Conclusions

We have developed genetically engineered E. aerogenes as a 2,3-butanediol producer that efficiently utilizes sugarcane molasses. The fermentation efficiency was dramatically improved by the alleviation of CCR and modulation of carbon preference. These results offer a metabolic engineering approach for achieving highly efficient utilization of mixed sugars for the biorefinery industry.

【 授权许可】

   
2015 Jung et al.

【 图 表 】

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【 参考文献 】

• [1]Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, et al.: The path forward for biofuels and biomaterials. Science 2006, 311:484-489.
• [2]van Haveren J, Scott EL, Sanders J: Bulk chemicals from biomass. Biofuel Bioprod Biorefin 2008, 2:41-57.
• [3]Ji XJ, Huang H, Ouyang PK: Microbial 2,3-butanediol production: a state-of-the-art review. Biotechnol Adv 2011, 29:351-364.
• [4]Celinska E, Grajek W: Biotechnological production of 2,3-butanediol—current state and prospects. Biotechnol Adv 2009, 27:715-725.
• [5]Wang AL, Wang Y, Jiang TY, Li LX, Ma CQ, Xu P: Production of 2,3-butanediol from corncob molasses, a waste by-product in xylitol production. Appl Microbiol Biotechnol 2010, 87:965-970.
• [6]Jiang LQ, Fang Z, Guo F, Yang LB: Production of 2,3-butanediol from acid hydrolysates of Jatropha hulls with Klebsiella oxytoca. Bioresour Technol 2012, 107:405-410.
• [7]Sun LH, Wang XD, Dai JY, Xiu ZL: Microbial production of 2,3-butanediol from Jerusalem artichoke tubers by Klebsiella pneumoniae. Appl Microbiol Biotechnol 2009, 82:847-852.
• [8]Dai JY, Zhao P, Cheng XL, Xiu ZL: Enhanced production of 2,3-butanediol from sugarcane molasses. Appl Biochem Biotechnol 2015, 175:3014-3024.
• [9]Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, et al.: Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005, 96:673-686.
• [10]Gao L, Chi Z, Sheng J, Ni X, Wang L: Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast Cryptococcus aureus G7a and its nutritive analysis. Appl Microbiol Biotechnol 2007, 77:825-832.
• [11]Goerke B, Stulke J: Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 2008, 6:613-624.
• [12]Deutscher J: The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol 2008, 11:87-93.
• [13]Zhang ZG, Aboulwafa M, Saier MH: Regulation of crp gene expression by the catabolite repressor/activator, Cra, in Escherichia coli. J Mol Microbiol Biotechnol 2014, 24:135-141.
• [14]Plumbridge J: Control of the expression of the manXYZ operon in Escherichia coli: Mlc is a negative regulator of the mannose PTS. Mol Microbiol 1998, 27:369-380.
• [15]Newman JR, Fuqua C: Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 1999, 227:197-203.
• [16]Shimada T, Yamamoto K, Ishihama A: Novel members of the Cra regulon involved in carbon metabolism in Escherichia coli. J Bacteriol 2011, 193:649-659.
• [17]Gawand P, Hyland P, Ekins A, Martin VJJ, Mahadevan R: Novel approach to engineer strains for simultaneous sugar utilization. Metab Eng 2013, 20:63-72.
• [18]Akaraonye E, Moreno C, Knowles JC, Keshavarz T, Roy I: Poly(3-hydroxybutyrate) production by Bacillus cereus SPV using sugarcane molasses as the main carbon source. Biotechnol J 2012, 7:293-303.
• [19]Jung MY, Park BS, Lee J, Oh MK: Engineered Enterobacter aerogenes for efficient utilization of sugarcane molasses in 2,3-butanediol production. Bioresour Technol 2013, 139:21-27.
• [20]Sprenger GA, Lengeler JW: Analysis of sucrose catabolism in Klebsiella-pneumoniae and in Scr+ derivatives of Escherichia coli K12. J Gen Microbiol 1988, 134:1635-1644.
• [21]Kornberg HL: Routes for fructose utilization by Escherichia coli. J Mol Microbiol Biotechechnol 2001, 3:355-359.
• [22]Deutscher J, Francke C, Postma PW: How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 2006, 70:939-1031.
• [23]Kelker NE, Hanson TE, Anderson RL: Alternate pathways of D-fructose metabolism in Aerobacter aerogenes—a specific D-fructokinase and its preferential role in metabolism of sucrose. J Biol Chem 1970, 245:2060-2065.
• [24]Saier MH, Ramseier TM: The catabolite repressor/activator (Cra) protein of enteric bacteria. J Bacteriol 1996, 178:3411-3417.
• [25]Yimga MT, Leatham MP, Allen JH, Laux DC, Conway T, Cohen PS: Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Tyhimurium in BALB/c mice. Infect Immun 2006, 74:1130-1140.
• [26]Ramseier TM, Bledig S, Michotey V, Feghali R, Saier MH: The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli. Mol Microbiol 1995, 16:1157-1169.
• [27]Jung MY, Ng CY, Song H, Lee J, Oh MK: Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production. Appl Microbiol Biotechnol 2012, 95:461-469.
• [28]Diancourt L, Passet V, Verhoef J, Grimont PAD, Brisse S: Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 2005, 43:4178-4182.
• [29]Reid SJ, Abratt VR: Sucrose utilisation in bacteria: genetic organisation and regulation. Appl Microbiol Biotechnol 2005, 67:312-321.
• [30]Ebner R, Lengeler JW: DNA-sequence of the gene scrA encoding the sucrose transport protein Enzymell scr of the phosphotransferase system from enteric bacteria—homology of the Enzymell scr and Enzymell bgl Proteins. Mol Microbiol 1988, 2:9-17.
• [31]Jahreis K, Lengeler JW: Molecular analysis of 2 ScrR repressors and of a ScrR-FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria. Mol Microbiol 1993, 9:195-209.
• [32]Ji XJ, Nie ZK, Huang H, Ren LJ, Peng C, Ouyang PK: Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose-xylose mixtures. Appl Microbiol Biotechnol 2011, 89:1119-1125.
• [33]Plumbridge J: Expression of ptsG, the gene for the major glucose PTS transporter in Escherichia coli, is repressed by MIc and induced by growth on glucose. Mol Microbiol 1998, 29:1053-1063.
• [34]Kimata K, Tanaka Y, Inada T, Aiba H: Expression of the glucose transporter gene, ptsG, is regulated at the mRNA degradation step in response to glycolytic flux in Escherichia coli. EMBO J 2001, 20:3587-3595.
• [35]Fernando S, Adhikari S, Chandrapal C, Murali N: Biorefineries: current status, challenges, and future direction. Energy Fuel 2006, 20:1727-1737.
• [36]Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, et al.: Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 2007, 315:804-807.
• [37]Cherubini F: The biorefinery concept: using biomass instead of oil for producing energy and chemicals. Energy Convers Manag 2010, 51:1412-1421.
• [38]Kim SR, Ha SJ, Wei N, Oh EJ, Jin YS: Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol. Trends Biotechnol 2012, 30:274-282.
• [39]Shin SH, Kim S, Kim JY, Lee S, Um Y, Oh MK, et al.: Complete genome sequence of Enterobacter aerogenes KCTC 2190. J Bacteriol 2012, 194:2373-2374.
• [40]Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000, 97:6640-6645.
• [41]Jung MY, Mazumdar S, Shin SH, Yang KS, Lee J, Oh MK: Improvement of 2,3-butanediol yield in Klebsiella pneumoniae by deletion of the pyruvate formate-lyase gene. Appl Environ Microbiol 2014, 80:6195-6203.