Abstract
Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F1-ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F1-ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F1-ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F1-ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished.
Similar content being viewed by others
References
Causey TB, Zhou S, Shanmugam KT, Ingram LO (2003) Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production. Proc Natl Acad Sci USA 100:825–832
De Carvalho CC (2011) Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv 29:75–83
Goldberg K, Schroer K, Lütz S, Liese A (2007) Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part II: whole-cell reductions. Appl Microbiol Biotechnol 76:249–255
Hugenholtz J (2008) The lactic acid bacterium as a cell factory for food ingredient production. Int Dairy J 18:466–475
Hädicke O, Bettenbrock K, Klamt S (2015) Enforced ATP futile cycling increases specific productivity and yield of anaerobic lactate production in Escherichia coli. Biotechnol Bioeng 112:2195–2199
Israelsen H, Madsen SM, Vrang A, EB H, Johansen E (1995) Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ Microbiol 61:2540–2547
Jensen P, Hammer K (1993) Minimal requirements for exponential growth of Lactococcus lactis. Appl Environ Microbiol 59:4363–4366
Kasjet ER (1987) Bioenergetics of lactic acid bacteria: citoplasmic pH and osmotolerance. FEMS Microbiol Rev 46:233–244
Kemp RG, Foe LG (1983) Allosteric regulatory properties of muscle phosphofructokinase. Mol Cell Biochem 57:147–154
Kim H-J, Kwon YD, Lee SY, Kim P (2012) An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production. Appl Microbiol Biotechnol 94:1079–1086
Koebmann BJ, Solem C, Pedersen MB, Nilsson D, Jensen PR (2002a) Expression of genes encoding F1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis. Appl Environ Microbiol 68:4274–4282
Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR (2002b) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. Appl Environ Microbiol 184:3909–3916
Lan EI, Liao JC (2012) ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc Natl Acad Sci USA 109:6018–6023
Lee SY, Kim HU (2015) Systems strategies for developing industrial microbial strains. Nat Biotechnol 33:1061–1072
Liang L, Liu R, Li F, Wu M, Chen K, Ma J, Jiang M, Wei P, Ouyang P (2013) Repetitive succinic acid production from lignocellulose hydrolysates by enhancement of ATP supply in metabolically engineered Escherichia coli. Bioresour Technol 143:405–412
Liu J, Chan SHJ, Brock-Nannestad T, Chen J, Lee SY, Solem C, Jensen PR (2016a) Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol. Metab Eng 36:57–67
Liu J, Dantoft SH, Würtz A, Jensen PR, Solem C (2016b) A novel cell factory for efficient production of ethanol from dairy waste. Biotechnol Biofuels 9:33
Liu R, Liang L, Chen K, Ma J, Jiang M, Wei P, Ouyang P (2012) Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 94:959–968
Matsumoto K, Taguchi S (2010) Enzymatic and whole-cell synthesis of lactate-containing polyesters: toward the complete biological production of polylactate. Appl Microbiol Biotechnol 85:921–932
Mijakovic I, Petranovic D, Jensen PR (2005) Tunable promoters in systems biology. Curr Opin Biotechnol 16:329–335
Park JH, Lee KH, Kim TY, Lee SY (2007) Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc Natl Acad Sci U S A 104:7797–7802
Patnaik R, Roof WD, Young RF, Liao JC (1992) Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle. J Bacteriol 174:7527–7532
Singh A, Cher Soh K, Hatzimanikatis V, Gill RT (2011) Manipulating redox and ATP balancing for improved production of succinate in E. coli. Metab Eng 13:76–81
Solem C, Defoor E, Jensen PR, Martinussen J (2008) Plasmid pCS1966, a new selection/counterselection tool for lactic acid bacterium strain construction based on the oroP gene, encoding an orotate transporter from Lactococcus lactis. Appl Environ Microbiol 74:4772–4775
Solem C, Dehli T, Jensen PR (2013) Rewiring Lactococcus lactis for ethanol production. Appl Environ Microbiol 79:2512–2518
Stephanopoulos G (2007) Challenges in engineering microbes for biofuels production. Science 315:801–804
Werpy T, Petersen G (2004) Top value added chemicals from biomass volume I—results of screening for potential candidates from sugars and synthesis gas. National renewable energy laboratory, Oak Ridge, TN
Teusink B, Smid EJ (2006) Modelling strategies for the industrial exploitation of lactic acid bacteria. Nat Rev Microbiol 4:46–56
Venayak N, Anesiadis N, Cluett WR, Mahadevan R (2015) Engineering metabolism through dynamic control. Curr Opin Biotechnol 34:142–152
Wegmann U, Overweg K, Jeanson S, Gasson M, Shearman C (2012) Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712. Microbiology 158:2936–2945
Xiao Z, Lu JR (2014) Strategies for enhancing fermentative production of acetoin: a review. Biotechnol Adv 32:492–503
Zhou J, Liu L, Shi Z, Du G, Chen J (2009) ATP in current biotechnology: regulation, applications and perspectives. Biotechnol Adv 27:94–101
Acknowledgments
This work was partly supported by a grant from the Innovation fund Denmark (4106-00037B).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
The article does not contain any studies with animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
Liu, J., Kandasamy, V., Würtz, A. et al. Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand. Appl Microbiol Biotechnol 100, 9509–9517 (2016). https://doi.org/10.1007/s00253-016-7687-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-016-7687-1