Leuconostoc mesenteroides subsp. cremoris is an obligate heterolactic fermentative lactic acid bacterium that is mostly used in industrial dairy fermentations. The phosphoketolase pathway (PKP) is a unique feature of the obligate heterolactic fermentation, which leads to the production of lactate, ethanol, and/or acetate, and the final product profile of PKP highly depends on the energetics and redox state of the organism. Another characteristic of the L. mesenteroides subsp. cremoris is the production of aroma compounds in dairy fermentation, such as in cheese production, through the utilization of citrate. Considering its importance in dairy fermentation, a detailed metabolic characterization of the organism is necessary for its more efficient use in the industry. To this aim, a genome-scale metabolic model of dairy-origin L. mesenteroides subsp. cremoris ATCC 19254 (iLM.c559) was reconstructed to explain the energetics and redox state mechanisms of the organism in full detail. The model includes 559 genes governing 1088 reactions between 1129 metabolites, and the reactions cover citrate utilization and citrate-related flavor metabolism. The model was validated by simulating co-metabolism of glucose and citrate and comparing the in silico results to our experimental results. Model simulations further showed that, in co-metabolism of citrate and glucose, no flavor compounds were produced when citrate could stimulate the formation of biomass. Significant amounts of flavor metabolites (e.g., diacetyl and acetoin) were only produced when citrate could not enhance growth, which suggests that flavor formation only occurs under carbon and ATP excess. The effects of aerobic conditions and different carbon sources on product profiles and growth were also investigated using the reconstructed model. The analyses provided further insights for the growth stimulation and flavor formation mechanisms of the organism.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Adler P, Bolten CJ, Dohnt K, Hansen CE, Wittmann C (2013) Core fluxome and metafluxome of lactic acid bacteria under simulated cocoa pulp fermentation conditions. Appl Environ Microbiol 79(18):5670–5681. https://doi.org/10.1128/AEM.01483-13
Bachmann H, Starrenburg MJ, Molenaar D, Kleerebezem M, van Hylckama Vlieg JE (2012) Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution. Genome Res 22(1):115–124. https://doi.org/10.1101/gr.121285.111
Bang J, Li L, Seong H, Kwon YW, Lee DY, Han NS (2017) Macromolecular and elemental composition analyses of Leuconostoc mesenteroides ATCC 8293 cultured in a chemostat. J Microbiol Biotechnol 27(5):939–942. https://doi.org/10.4014/jmb.1612.12038
Bourel G, Henini S, Divies C, Garmyn D (2003) The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas. J Appl Microbiol 94(2):280–288
Campedelli I, Flórez AB, Salvetti E, Delgado S, Orrù L, Cattivelli L, Alegría Á, Felis GE, Torriani S, Mayo B (2015) Draft genome sequence of three antibiotic-resistant Leuconostoc mesenteroides strains of dairy origin. Genome Announc 3(5):e01018–e01015
Carroll AL, Desai SH, Atsumi S (2016) Microbial production of scent and flavor compounds. Curr Opin Biotechnol 37:8–15. https://doi.org/10.1016/j.copbio.2015.09.003
Christiaens JF, Franco LM, Cools TL, De Meester L, Michiels J, Wenseleers T, Hassan BA, Yaksi E, Verstrepen KJ (2014) The fungal aroma gene ATF1 promotes dispersal of yeast cells through insect vectors. Cell Rep 9(2):425–432. https://doi.org/10.1016/j.celrep.2014.09.009
Chun BH, Kim KH, Jeon HH, Lee SH, Jeon CO (2017) Pan-genomic and transcriptomic analyses of Leuconostoc mesenteroides provide insights into its genomic and metabolic features and roles in kimchi fermentation. Sci Rep 7(1):11504. https://doi.org/10.1038/s41598-017-12,016-z
Cogan TM, Beresford TP, Steele J, Broadbent J, Shah NP, Ustunol Z (2007) Invited review: advances in starter cultures and cultured foods. J Dairy Sci 90(9):4005–4021. https://doi.org/10.3168/jds.2006-765
Cosgrove MS, Naylor C, Paludan S, Adams MJ, Levy HR (1998) On the mechanism of the reaction catalyzed by glucose 6-phosphate dehydrogenase. Biochemistry 37(9):2759–2767. https://doi.org/10.1021/bi972069y
de Paula AT, Jeronymo-Ceneviva AB, Todorov SD, Penna ALB (2015) The two faces of Leuconostoc mesenteroides in food systems. Food Rev Int 31(2):147–171. https://doi.org/10.1080/87559129.2014.981825
Dols M, Chraibi W, Remaud-Simeon M, Lindley ND, Monsan PF (1997) Growth and energetics of Leuconostoc mesenteroides NRRL B-1299 during metabolism of various sugars and their consequences for dextransucrase production. Appl Environ Microbiol 63(6):2159–2165
Flahaut NAL, Wiersma A, van de Bunt B, Martens DE, Schaap PJ, Sijtsma L, dos Santos VAM, de Vos WM (2013) Genome-scale metabolic model for Lactococcus lactis MG1363 and its application to the analysis of flavor formation. Appl Microbiol Biotechnol 97(19):8729–8739. https://doi.org/10.1007/s00253-013-5140-2
Frantzen CA, Kot W, Pedersen TB, Ardo YM, Broadbent JR, Neve H, Hansen LH, Dal Bello F, Ostlie HM, Kleppen HP, Vogensen FK, Holo H (2017) Genomic characterization of dairy associated Leuconostoc species and diversity of Leuconostocs in undefined mixed mesophilic starter cultures. Front Microbiol 8:132. https://doi.org/10.3389/fmicb.2017.00132
Ganzle MG (2015) Lactic metabolism revisited: metabolism of lactic acid bacteria in food fermentations and food spoilage. Curr Opin Food Sci 2:106–117. https://doi.org/10.1016/j.cofs.2015.03.001
Garvie EI (1986) Genus Leuconostoc. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, 2nd edn. Springer-Verlag, New York
Gaspar P, Carvalho AL, Vinga S, Santos H, Neves AR (2013) From physiology to systems metabolic engineering for the production of biochemicals by lactic acid bacteria. Biotechnol Adv 31(6):764–788. https://doi.org/10.1016/j.biotechadv.2013.03.011
Gunsalus IC, Gibbs M (1952) The heterolactic fermentation. II. Position of C14 in the products of glucose dissimilation by Leuconostoc mesenteroides. J Biol Chem 194(2):871–875
Guzel-Seydim ZB, Kok-Tas T, Greene AK, Seydim AC (2011) Review: functional properties of kefir. Crit Rev Food Sci Nutr 51(3):261–268. https://doi.org/10.1080/10408390903579029
Harney SJ, Simopoulos ND, Ikawa M (1967) Cell wall constituents of Leuconostoc citrovorum and Leuconostoc mesenteroides. J Bacteriol 93(1):273–277
Hemme D, Foucaud-Scheunemann C (2004) Leuconostoc, characteristics, use in dairy technology and prospects in functional foods. Int Dairy J 14(6):467–494. https://doi.org/10.1016/j.idairyj.2003.10.005
Henry CS, DeJongh M, Best AA, Frybarger PM, Linsay B, Stevens RL (2010) High-throughput generation, optimization and analysis of genome-scale metabolic models. Nat Biotechnol 28(9):977–982. https://doi.org/10.1038/nbt.1672
Human Microbiome Project C (2012a) A framework for human microbiome research. Nature 486(7402):215–221. https://doi.org/10.1038/nature11209
Human Microbiome Project C (2012b) Structure, function and diversity of the healthy human microbiome. Nature 486(7402):207–214. https://doi.org/10.1038/nature11234
Koduru L, Kim Y, Bang J, Lakshmanan M, Han NS, Lee DY (2017) Genome-scale modeling and transcriptome analysis of Leuconostoc mesenteroides unravel the redox governed metabolic states in obligate heterofermentative lactic acid bacteria. Sci Rep 7(1):15721. https://doi.org/10.1038/s41598-017-16026-9
Konings WN (2002) The cell membrane and the struggle for life of lactic acid bacteria. Antonie Van Leeuwenhoek 82(1-4):3–27
Leroy F, De Vuyst L (2004) Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol 15(2):67–78. https://doi.org/10.1016/j.tifs.2003.09.004
LevataJovanovic M, Sandine WE (1996) Citrate utilization and diacetyl production by various strains of Leuconostoc mesenteroides ssp cremoris. J Dairy Sci 79(11):1928–1935. https://doi.org/10.3168/jds.S0022-0302(96)76562-1
Levy HR (1989) Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Biochem Soc Trans 17(2):313–315
Levy HR, Christoff M, Ingulli J, Ho EM (1983) Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition. Arch Biochem Biophys 222(2):473–488
Mahadevan R, Schilling CH (2003) The effects of alternate optimal solutions in constraint-based genome-scale metabolic models. Metab Eng 5(4):264–276
Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31(3):426–428. https://doi.org/10.1021/ac60147a030
Naylor CE, Gover S, Basak AK, Cosgrove MS, Levy HR, Adams MJ (2001) NADP+ and NAD+ binding to the dual coenzyme specific enzyme Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: different interdomain hinge angles are seen in different binary and ternary complexes. Acta Crystallogr D Biol Crystallogr 57(Pt 5):635–648
Nielsen J (2017) Systems biology of metabolism. Annu Rev Biochem 86:245–275. https://doi.org/10.1146/annurev-biochem-061516-044757
Olive C, Geroch ME, Levy HR (1971) Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides. Kinetic studies. J Biol Chem 246(7):2047–2057
Oliveira AP, Nielsen J, Forster J (2005) Modeling Lactococcus lactis using a genome-scale flux model. Bmc Microbiol 5:39. https://doi.org/10.1186/1471-2180-5-39
Orth JD, Thiele I, Palsson BO (2010) What is flux balance analysis? Nat Biotechnol 28(3):245–248. https://doi.org/10.1038/nbt.1614
Otto R, Tenbrink B, Veldkamp H, Konings WN (1983) The relation between growth-rate and electrochemical proton gradient of Streptococcus cremoris. FEMS Microbiol Lett 16(1):69–74
Pastink MI, Teusink B, Hols P, Visser S, de Vos WM, Hugenholtz J (2009) Genome-scale model of Streptococcus thermophilus LMG18311 for metabolic comparison of lactic acid bacteria. Appl Environ Microbiol 75(11):3627–3633. https://doi.org/10.1128/AEM.00138-09
Pedersen MB, Gaudu P, Lechardeur D, Petit MA, Gruss A (2012) Aerobic respiration metabolism in lactic acid bacteria and uses in biotechnology. Annu Rev Food Sci Technol 3:37–58. https://doi.org/10.1146/annurev-food-022811-101,255
Plihon F, Taillandier P, Strehaiano P (1995) Oxygen effect on batch cultures of Leuconostoc mesenteroides - relationship between oxygen-uptake, growth and end-products. Appl Microbiol Biotechnol 43(1):117–122
Poolman B, Konings WN (1988) Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino-acid transport. J Bacteriol 170(2):700–707
Schellenberger J, Que R, Fleming RM, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BO (2011) Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nat Protoc 6(9):1290–1307. https://doi.org/10.1038/nprot.2011.308
Schmitt P, Divies C (1992) Effect of varying citrate levels on C-4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp cremoris grown in continuous culture. Appl Microbiol Biotechnol 37(4):426–430
Schmitt P, Divies C, Cardona R (1992) Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp cremoris. Appl Microbiol Biotechnol 36(5):679–683
Shi Z, Li H, Li Z, Hu J, Zhang H (2013) Pre-column derivatization RP-HPLC determination of amino acids in Asparagi radix before and after heating process. IERI Procedia 5:351–356. https://doi.org/10.1016/j.ieri.2013.11.115
Smid EJ, Erkus O, Spus M, Wolkers-Rooijackers JCM, Alexeeva S, Kleerebezem M (2014) Functional implications of the microbial community structure of undefined mesophilic starter cultures. Microb Cell Fact 13:S2. https://doi.org/10.1186/1475-2859-13-S1-S2
Starrenburg MJC, Hugenholtz J (1991) Citrate fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol 57(12):3535–3540
Teusink B, van Enckevort FH, Francke C, Wiersma A, Wegkamp A, Smid EJ, Siezen RJ (2005) In silico reconstruction of the metabolic pathways of Lactobacillus plantarum: comparing predictions of nutrient requirements with those from growth experiments. Appl Environ Microbiol 71(11):7253–7262. https://doi.org/10.1128/AEM.71.11.7253-7262.2005
Teusink B, Wiersma A, Molenaar D, Francke C, de Vos WM, Siezen RJ, Smid EJ (2006) Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model. J Biol Chem 281(52):40041–40048. https://doi.org/10.1074/jbc.M606263200
Tracey RP, Britz TJ (1989) Cellular fatty-acid composition of Leuconostoc oenos. J Appl Bacteriol 66(5):445–456. https://doi.org/10.1111/j.1365-2672.1989.tb05114.x
van Mastrigt O, Abee T, Lillevang SK, Smid EJ (2018) Quantitative physiology and aroma formation of a dairy Lactococcus lactis at near-zero growth rates. Food Microbiol 73:216–226. https://doi.org/10.1016/j.fm.2018.01.027
Vinay-Lara E, Hamilton JJ, Stahl B, Broadbent JR, Reed JL, Steele JL (2014) Genome-scale reconstruction of metabolic networks of Lactobacillus casei ATCC 334 and 12A. PLoS One 9(11):e110785. https://doi.org/10.1371/journal.pone.0110785
Yi YJ, Lim JM, Gu S, Lee WK, Oh E, Lee SM, Oh BT (2017) Potential use of lactic acid bacteria Leuconostoc mesenteroides as a probiotic for the removal of Pb(II) toxicity. J Microbiol 55(4):296–303. https://doi.org/10.1007/s12275-017-6642-x
Burcu Şirin (Yeditepe University) and Sebastián N. Mendoza (VU Amsterdam) are acknowledged for their help in HPLC analyses and reorganizing the SBML file, respectively.
This work received financial support from The Scientific and Technological Research Council of Turkey through TUBITAK 2214-A program and the Marmara University Scientific Research Project Fund through Project No: FEN-C-DRP-091116-0498.
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Özcan, E., Selvi, S.S., Nikerel, E. et al. A genome-scale metabolic network of the aroma bacterium Leuconostoc mesenteroides subsp. cremoris. Appl Microbiol Biotechnol 103, 3153–3165 (2019). https://doi.org/10.1007/s00253-019-09630-4
- Lactic acid bacteria
- Leuconostoc mesenteroides subsp. cremoris
- Heterolactic fermentation
- Flavor metabolism
- Genome-scale metabolic model
- Flux balance analysis