Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

Biotechnology for Biofuels

Xiao-Jun Ji¹  Zhi-Fang Xia¹  Ning-Hua Fu¹  Zhi-Kui Nie¹  Meng-Qiu Shen¹  Qian-Qian Tian¹  He Huang¹ 

[1] State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing, 210009, People’s Republic of China

关键词: NADH oxidase;    Klebsiella pneumoniae;    Cofactor engineering;    2,3-Butanediol;    Acetoin;   

Others  :  798179
DOI  :  10.1186/1754-6834-6-7

 received in 2012-12-19, accepted in 2013-01-23,  发布年份 2013

【 摘 要 】

Background

Acetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria.

Results

The present study reports introducing an exogenous NAD⁺ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold) and NADH/NAD⁺ ratio (2.0 fold). Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L) by Klebsiella species was obtained.

Conclusions

The present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD⁺ ratio in K. pneumoniae. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD⁺-dependent microbial metabolite.

【 授权许可】

   
2013 Ji et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]San KY, Bennett GN, Berríos-Rivera SJ, Vadali RV, Yang YT, Horton E, Rudolph FB, Sariyar B, Blackwood K: Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metab Eng 2002, 4:182-192.
• [2]Liu LM, Chen J: Cofactor engineering enhances the physiological function of an industrial strain. In Progress in Molecular and Environmental Bioengineering–From Analysis and Modeling to Technology Applications. Edited by Carpi A. InTech Open Access Publisher, Rijeka; 2011:427-444.
• [3]Jang YS, Park JM, Choi S, Choi YJ, Seung DY, Cho JH, Lee SY: Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnol Adv 2011, 30:989-1000.
• [4]de Felipe FL, Kleerebezem M, de Vos WM, Hugenholtz J: Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J Bacteriol 1998, 180:3804-3808.
• [5]Foster JW, Park YK, Penfound T, Fenger T, Spector MP: Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon. J Bacteriol 1990, 172:4187-4196.
• [6]Heux S, Cachon R, Dequin S: Cofactor engineering in Saccharomyces cerevisiae: expression of a H2O-forming NADH oxidase and impact on redox metabolism. Metab Eng 2006, 8:303-314.
• [7]Berríos-Rivera SJ, San KY, Bennett GN: The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli. J Ind Microbiol Biotechnol 2003, 30:34-40.
• [8]Ji XJ, Huang H, Zhu JG, Ren LJ, Nie ZK, Du J, Li S: Engineering Klebsiella oxytoca for efficient 2,3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene. Appl Microbiol Biotechnol 2010, 85:1751-1758.
• [9]Zhang YP, Li Y, Du CY, Liu M, Cao ZA: Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae. Metab Eng 2006, 8:578-586.
• [10]Berríos-Rivera SJ, Bennett GN, San KY: Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metab Eng 2002, 4:217-229.
• [11]Zhang YP, Huang ZH, Du CY, Li Y, Cao ZA: Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol. Metab Eng 2009, 11:101-106.
• [12]Berríos-Rivera SJ, San KY, Bennett GN: The effect of NAPRTase overexpression on the total levels of NAD, the NADH/NAD+ ratio, and the distribution of metabolites in Escherichia coli. Metab Eng 2002, 4:238-247.
• [13]Liu LM, Li Y, Shi ZP, Du GC, Chen J: Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD+ availability. J Biotechnol 2006, 126:173-185.
• [14]Sánchez AM, Bennett GN, San KY: Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium. J Biotechnol 2005, 117:395-405.
• [15]Werpy T, Petersen G: Top value added chemicals from biomass, Volume 1: results of screening for potential candidates from sugars and synthesis gas. http://www1.eere.energy.gov/biomass/pdfs/35523.pdf webcite
• [16]Xiao ZJ, Xu P: Acetoin metabolism in bacteria. Crit Rev Microbiol 2007, 33:127-140.
• [17]Xiao Z, Wang X, Huang Y, Huo F, Zhu X, Xi L, Liu JR: Thermophilic fermentation of acetoin and 2,3-butanediol by a novel Geobacillus strain. Biotechnol Biofuels 2012, 5:88. BioMed Central Full Text
• [18]Ji XJ, Huang H, Nie ZK, Qu L, Xu Q, Tsao GT: Fuels and chemicals from hemicellulose sugars. Adv Biochem Eng Biotechnol 2012, 128:199-224.
• [19]Liu YF, Zhang SL, Yong YC, Ji ZX, Ma X, Xu ZH, Chen SW: Efficient production of acetoin by the newly isolated Bacillus licheniformis strain MEL09. Process Biochem 2011, 46:390-394.
• [20]Xu P, Xiao Z, Du Y, Wei Z: An acetoin high yield Bacillus pumilus strain. European Patent 2009,  .
• [21]Zhang X, Yang T, Lin Q, Xu M, Xia H, Xu Z, Li H, Rao Z: Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation. World J Microbiol Biotechnol 2011, 12:2785-2790.
• [22]Ji XJ, Huang H, Ouyang PK: Microbial 2,3-butanediol production: A state-of-the-art review. Biotechnol Adv 2011, 29:351-364.
• [23]Johansen L, Bryn K, Störmer FC: Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes. J Bacteriol 1975, 123:1124-1130.
• [24]Afschar AS, Bellgardt KH, Rossell CE, Czok A, Schaller K: The production of 2,3-butanediol by fermentation of high test molasses. Appl Microbiol Biotechnol 1991, 34:582-585.
• [25]Ji XJ, Huang H, Du J, Zhu JG, Ren LJ, Hu N, Li S: Enhanced 2,3-butanediol production by Klebsiella oxytoca using a two-stage agitation speed control strategy. Bioresour Technol 2009, 100:3410-3414.
• [26]Nie ZK, Ji XJ, Huang H, Du J, Li ZY, Qu L, Zhang Q, Ouyang PK: An effective and simplified fed-batch strategy for improved 2,3-butanediol production by Klebsiella oxytoca. Appl Biochem Biotechnol 2011, 163:946-953.
• [27]Qureshi N, Cheryan M: Production of 2,3-butanediol by Klebsiella oxytoca. Appl Microbiol Biotechnol 1989, 30:440-443.
• [28]Li D, Dai JY, Xiu ZL: A novel strategy for integrated utilization of Jerusalem artichoke stalk and tuber for production of 2,3-butanediol by Klebsiella pneumoniae. Bioresour Technol 2010, 101:8342-8347.
• [29]Ma CQ, Wang AL, Qin JY, Li LX, Ai XL, Jiang TY, Tang HZ, Xu P: Enhanced 2,3-butanediol production by Klebsiella pneumoniae SDM. Appl Microbiol Biotechnol 2009, 82:49-57.
• [30]Qin JY, Xiao ZJ, Ma CQ, Xie NZ, Liu PH, Xu P: Production of 2,3-butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate. Chin J Chem Eng 2006, 14:132-136.
• [31]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.
• [32]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.
• [33]Hou J, Lages NF, Oldiges M, Vemuri GN: Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae. Metab Eng 2009, 11:253-261.
• [34]Vemuri G, Eiteman M, McEwen J, Olsson L, Nielsen J: Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 2007, 104:2402-2407.
• [35]Fournet-Fayard S, Joly B, Forestier C: Transformation of wild type Klebsiella pneumoniae with plasmid DNA by electroporation. J Microbiol Methods 1995, 24:49-54.
• [36]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.
• [37]Zhu JG, Li S, Ji XJ, Huang H, Hu N: Enhanced 1,3-propanediol production in recombinant Klebsiella pneumoniae carrying the gene yqhD encoding 1,3-propanediol oxidoreductase isoenzyme. World J Microbiol Biotechnol 2009, 25:1217-1223.
• [38]Auzat I, Chapuy-Regaud S, Bras GL, Santos DD, Ogunniyi AD, Thomas IL, Garel JR, Paton JC, Trombe MC: The NADH oxidase of Streptococcus pneumoniae: its involvement incompetence and virulence. Mol Microbiol 1999, 34:1018-1028.
• [39]Kleiner D, Paul W, Merrick MJ: Construction of multicopy expreesion vectors for regulated overproduction of proteins in Klebsiella pneumoniae and other enteric bacteria. J Gen Microbiol 1988, 134:1779-1784.
• [40]Joseph S, David WR: Molecular cloning: a laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press, New York; 2001.
• [41]de Felipe FL, Hugenholtz J: Purification and characterisation of the water forming NADH-oxidase from Lactococcus lactis. Intern Dairy J 2001, 11:37-44.
• [42]Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254.
• [43]Lilius EM, Multanen VM, Toivonen V: Quantitative extraction and estimation of intracellular nicotinamid dinucleotides in Escherichia coli. Anal Biochem 1979, 99:22-27.
• [44]Bernowsky C, Swan M: An improved cycling assay for nicotinamide adenine dinucleotide. Anal Biochem 1973, 53:452-458.