Browse > Article
http://dx.doi.org/10.4014/jmb.1801.01045

Analysis of the Growth and Metabolites of a Pyruvate Dehydrogenase Complex-Deficient Klebsiella pneumoniae Mutant in a Glycerol-Based Medium  

Xu, Danfeng (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Jia, Zongxiao (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Zhang, Lijuan (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Fu, Shuilin (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Gong, Heng (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Publication Information
Journal of Microbiology and Biotechnology / v.30, no.5, 2020 , pp. 753-761 More about this Journal
Abstract
To determine the role of pyruvate dehydrogenase complex (PDHC) in Klebsiella pneumoniae, the growth and metabolism of PDHC-deficient mutant in glycerol-based medium were analyzed and compared with those of other strains. Under aerobic conditions, the PDHC activity was fourfold higher than that of pyruvate formate lyase (PFL), and blocking of PDHC caused severe growth defect and pyruvate accumulation, indicating that the carbon flux through pyruvate to acetyl coenzyme A mainly depended on PDHC. Under anaerobic conditions, although the PDHC activity was only 50% of that of PFL, blocking of PDHC resulted in more growth defect than blocking of PFL. Subsequently, combined with the requirement of CO2 and intracellular redox status, it was presumed that the critical role of PDHC was to provide NADH for the anaerobic growth of K. pneumoniae. This presumption was confirmed in the PDHC-deficient mutant by further blocking one of the formate dehydrogenases, FdnGHI. Besides, based on our data, it can also be suggested that an improvement in the carbon flux in the PFL-deficient mutant could be an effective strategy to construct high-yielding 1,3-propanediol-producing K. pneumoniae strain.
Keywords
1,3-propanediol; glycerol; Klebsiella pneumoniae; pyruvate dehydrogenase complex; pyruvate formate lyase;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Wilkinson KD, Williams CH, Jr. 1981. NADH inhibition and NAD activation of Escherichia coli lipoamide dehydrogenase catalyzing the NADH-lipoamide reaction. J. Biol. Chem. 256: 2307-2314.   DOI
2 Murarka A, Clomburg JM, Moran S, Shanks JV, Gonzalez R. 2010. Metabolic analysis of wild-type Escherichia coli and a pyruvate dehydrogenase complex (PDHC)-deficient derivative reveals the role of PDHC in the fermentative metabolism of glucose. J. Biol. Chem. 285: 31548-31558.   DOI
3 Menzel K, Ahrens K, Zeng A, Deckwer W. 1998. Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism. Biotechnol. Bioeng. 60: 617-626.   DOI
4 Menzel K, Zeng AP, Deckwer WD. 1997. Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J. Biotechnol. 56: 135-142.   DOI
5 Yamamoto S, Izumiya H, Morita M, Arakawa E, Watanabe H. 2009. Application of lambda red recombination system to Vibrio cholerae genetics: simple methods for inactivation and modification of chromosomal genes. Gene 438: 57-64.   DOI
6 Garrigues C, Loubiere P, Lindley ND, Cocaign-Bousquet M. 1997. Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the $NADH/NAD^+$ ratio. J. Bacteriol. 179: 5282-5287.   DOI
7 Russell GC, Guest JR. 1990. Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue. Biochem. J. 269: 443-450.   DOI
8 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: 248-254.   DOI
9 Teng WK, Ngoh GC, Yusoff R, Aroua MK. 2016. Microwave-assisted transesterification of industrial grade crude glycerol for the production of glycerol carbonate. Chem. Eng. J. 284: 469-477.   DOI
10 Dobson R, Gray V, Rumbold K. 2012. Microbial utilization of crude glycerol for the production of value-added products. J. Ind. Microbiol. Biotechnol 39: 217-226.   DOI
11 Quispe CAG, Coronado CJR, Carvalho JA. 2013. Glycerol: Production, consumption, prices, characterization and new trends in combustion. Renew. Sustain. Energy Rev. 27: 475-493.   DOI
12 Xiu ZL, Zeng AP. 2008. Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl. Microbiol. Biotechnol. 78: 917-926.   DOI
13 Xu G, Liu Y, Gao Q. 2016. Multi-objective optimization of a continuous bio-dissimilation process of glycerol to 1, 3-propanediol. J. Biotechnol. 219: 59-71.   DOI
14 Eikmanns BJ, Blombach B. 2014. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering. J. Biotechnol. 192 Pt B: 339-345.   DOI
15 Zhang YM, Luo JA, Zhao XB, Liu DH. 2015. A novel strategy for 1,3-propanediol recovery from fermentation broth and control of product colority using scraped thin-film evaporation for desalination. RSC Adv. 5: 48269-48274.   DOI
16 Gungormusler-Yilmaz M, Cicek N, Levin DB, Azbar N. 2016. Cell immobilization for microbial production of 1,3-propanediol. Crit. Rev. Biotechnol. 36: 482-494.
17 Chen T, Liu WX, Fu J, Zhang B, Tang YJ. 2013. Engineering Bacillus subtilis for acetoin production from glucose and xylose mixtures. J. Biotechnol. 168: 499-505.   DOI
18 Wang Y, Tao F, Xin B, Liu H, Gao Y, Zhou NY, et al. 2017. Switch of metabolic status: redirecting metabolic flux for acetoin production from glycerol by activating a silent glycerol catabolism pathway. Metab. Eng. 39: 90-101.   DOI
19 Samuelov NS, Lamed R, Lowe S, Zeikus JG. 1991. Influence of $CO_2-HCO_3$ levels and pH on growth, succinate production, and enzyme activities of Anaerobiospirillum succiniciproducens. Appl. Environ. Microbiol. 57: 3013-3019.   DOI
20 Stephens PE, Darlison MG, Lewis HM, Guest JR. 1983. The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component. Eur. J. Biochem. 133: 481-489.   DOI
21 Skorokhodova AY, Morzhakova AA, Gulevich AY, Debabov VG. 2015. Manipulating pyruvate to acetyl-CoA conversion in Escherichia coli for anaerobic succinate biosynthesis from glucose with the yield close to the stoichiometric maximum. J. Biotechnol. 214: 33-42.   DOI
22 Alscher G, Krug H, Liebig HP. 2001. Optimisation of $CO_2$ and temperature control in greenhouse crops by means of growth models at different abstraction levels - I. Control strategies, growth models and input data. Gartenbauwissenschaft 66: 105-114.
23 Lin J, Zhang YQ, Xu DF, Xiang G, Jia ZX, Fu SL, et al. 2016. Deletion of poxB, pta, and ackA improves 1,3-propanediol production by Klebsiella pneumoniae. Appl. Microbiol. Biotechnol. 100: 2775-2784.   DOI
24 Hsu HH, Abbo BG. 2004. Role of bicarbonate/$CO^2$ buffer in the initiation of vesicle-mediated calcification: mechanisms of aortic calcification related to atherosclerosis. Biochim. Biophys. Acta 1690: 118-123.   DOI
25 Sawers RG, Blokesch M, Bock A. 2004. Anaerobic formate and hydrogen metabolism. EcoSal Plus. 1(1). doi: 10.1128/ecosalplus.3.5.4.
26 Beyer L, Doberenz C, Falke D, Hunger D, Suppmann B, Sawers RG. 2013. Coordination of FocA and pyruvate formate-lyase synthesis in Escherichia coli demonstrates preferential translocation of formate over other mixed-acid fermentation products. J. Bacteriol. 195: 1428-1435.   DOI
27 Gao LR, Jiang X, Fu SL, Gong H. 2014. In silico identification of potential virulence genes in 1,3-propanediol producer Klebsiella pneumonia. J. Biotechnol. 189: 9-14.   DOI
28 Huang YN, Li ZM, Shimizu K, Ye Q. 2012. Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae. Bioresour. Technol. 103: 351-359.   DOI
29 Zhu CQ, Jiang X, Zhang YQ, Lin J, Fu SL, Gong H. 2015. Improvement of 1,3-propanediol production in Klebsiella pneumoniae by moderate expression of puuC (encoding an aldehyde dehydrogenase). Biotechnol. Lett. 37: 1783-1790.   DOI
30 Cui YL, Zhou JJ, Gao LR, Zhu CQ, Jiang X, Fu SL, et al. 2014. Utilization of excess NADH in 2,3-butanediol-deficient Klebsiella pneumoniae for 1,3-propanediol production. J. Appl. Microbiol. 117: 690-698.   DOI
31 Xu YZ, Guo NN, Zheng ZM, Ou XJ, Liu HJ, Liu DH. 2009. Metabolism in 1,3-Propanediol Fed-Batch Fermentation by a D-Lactate Deficient Mutant of Klebsiella pneumoniae. Biotechnol. Bioeng. 104: 965-972.   DOI
32 Snoep JL, de Graef MR, Westphal AH, de Kok A, Teixeira de Mattos MJ, Neijssel OM. 1993. Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo. FEMS Microbiol. Lett. 114: 279-283.   DOI
33 Wang QZ, Ou MS, Kim Y, Ingram LO, Shanmugam KT. 2010. Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase. Appl. Environ. Microbiol. 76: 2107-2114.   DOI
34 Jung MY, Mazumdar S, Shin SH, Yang KS, Lee J, Oh MK. 2014. Improvement of 2,3-butanediol yield in Klebsiella pneumoniae by deletion of the pyruvate formate-lyase gene. Appl. Environ. Microbiol. 80: 6195-6203.   DOI
35 Thome R, Gust A, Toci R, Mendel R, Bittner F, Magalon A, et al. 2012. A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli. J. Biol. Chem. 287: 4671-4678.   DOI
36 Wu Z, Wang Z, Wang G, Tan T. 2013. Improved 1,3-propanediol production by engineering the 2,3-butanediol and formic acid pathways in integrative recombinant Klebsiella pneumoniae. J. Biotechnol. 168: 194-200.   DOI
37 Hasona A, Kim Y, Healy FG, Ingram LO, Shanmugam KT. 2004. Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose. J. Bacteriol. 186: 7593-7600.   DOI
38 Lim JH, Seo SW, Kim SY, Jung GY. 2013. Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli. Metab. Eng. 20: 49-55.   DOI
39 Zhang YP, Huang ZH, Du CY, Li Y, Cao ZA. 2009. Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol. Metab. Eng. 11: 101-106.   DOI
40 Singh A, Lynch MD, Gill RT. 2009. Genes restoring redox balance in fermentation-deficient E. coli NZN111. Metab. Eng. 11: 347-354.   DOI
41 Zhu J, Shimizu K. 2004. The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Appl. Microbiol. Biotechnol. 64: 367-375.   DOI
42 Bar-Even A. 2016. Formate assimilation: the metabolic architecture of natural and synthetic pathways. Biochem. 55: 3851-3863.   DOI