Metabolomic and proteomic analysis of d-lactate-producing Lactobacillus delbrueckii under various fermentation conditions
- 403 Downloads
As an important feedstock monomer for the production of biodegradable stereo-complex poly-lactic acid polymer, d-lactate has attracted much attention. To improve d-lactate production by microorganisms such as Lactobacillus delbrueckii, various fermentation conditions were performed, such as the employment of anaerobic fermentation, the utilization of more suitable neutralizing agents, and exploitation of alternative nitrogen sources. The highest d-lactate titer could reach 133 g/L under the optimally combined fermentation condition, increased by 70.5% compared with the control. To decipher the potential mechanisms of d-lactate overproduction, the time-series response of intracellular metabolism to different fermentation conditions was investigated by GC–MS and LC–MS/MS-based metabolomic analysis. Then the metabolomic datasets were subjected to weighted correlation network analysis (WGCNA), and nine distinct metabolic modules and eight hub metabolites were identified to be specifically associated with d-lactate production. Moreover, a quantitative iTRAQ–LC–MS/MS proteomic approach was employed to further analyze the change of intracellular metabolism under the combined fermentation condition, identifying 97 up-regulated and 42 down-regulated proteins compared with the control. The in-depth analysis elucidated how the key factors exerted influence on d-lactate biosynthesis. The results revealed that glycolysis and pentose phosphate pathways, transport of glucose, amino acids and peptides, amino acid metabolism, peptide hydrolysis, synthesis of nucleotides and proteins, and cell division were all strengthened, while ATP consumption for exporting proton, cell damage, metabolic burden caused by stress response, and bypass of pyruvate were decreased under the combined condition. These might be the main reasons for significantly improved d-lactate production. These findings provide the first omics view of cell growth and d-lactate overproduction in L. delbrueckii, which can be a theoretical basis for further improving the production of d-lactate.
Keywordsd-lactate Fermentation condition Metabolomics Proteomics Lactobacillus delbrueckii
This work was financially supported by the National Natural Science Foundation of China (no. 21676189); and the key technologies R & D program of Tianjin (no. 16YFZCSY00780).
- 1.Abdel-Banat BMA, Hoshida H, Ano A, Nonklang S, Akada R (2010) High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol 85:861–867. https://doi.org/10.1007/s00253-009-2248-5 CrossRefPubMedGoogle Scholar
- 5.Courant F, Martzolff A, Rabin G, Antignac J-P, Le Bizec B, Giraudeau P, Tea I, Akoka S, Couzinet A, Cogne G, Grizeau D, Goncalves O (2013) How metabolomics can contribute to bio-processes: a proof of concept study for biomarkers discovery in the context of nitrogen-starved microalgae grown in photobioreactors. Metabolomics 9:1286–1300. https://doi.org/10.1007/s11306-013-0532-y CrossRefGoogle Scholar
- 26.Koponen J, Laakso K, Koskenniemi K, Kankainen M, Savijoki K, Nyman TA, de Vos WM, Tynkkynen S, Kalkkinen N, Varmanen P (2012) Effect of acid stress on protein expression and phosphorylation in Lactobacillus rhamnosus GG. J Proteom 75:1357–1374. https://doi.org/10.1016/j.jprot.2011.11.009 CrossRefGoogle Scholar
- 29.Lv L-X, Yan R, Shi H-Y, Shi D, Fang D-Q, Jiang H-Y, Wu W-R, Guo F-F, Jiang X-W, Gu S-L, Chen Y-B, Yao J, Li L-J (2017) Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01. J Proteom 150:216–229. https://doi.org/10.1016/j.jprot.2016.08.021 CrossRefGoogle Scholar
- 30.Marty-Teysset C, de la Torre F, Garel JR (2000) Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress. Appl Environ Microbiol 66:262–267. https://doi.org/10.1128/aem.66.1.262-267.2000 CrossRefPubMedPubMedCentralGoogle Scholar
- 31.Mimitsuka T, Na K, Morita K, Sawai H, Minegishi S, Henmi M, Yamada K, Shimizu S, Yonehara T (2012) A membrane-integrated fermentation reactor system: its effects in reducing the amount of sub-raw materials for d-lactic acid continuous fermentation by Sporolactobacillus laevolacticus. Biosci Biotechnol Biochem 76:67–72. https://doi.org/10.1271/bbb.110499 CrossRefPubMedGoogle Scholar
- 34.Mura A, Fadda D, Perez AJ, Danforth ML, Musu D, Rico AI, Krupka M, Denapaite D, Tsui H-CT, Winkler ME, Branny P, Vicente M, Margolin W, Massidda O (2017) Roles of the essential protein FtsA in cell growth and division in Streptococcus pneumoniae. J Bacteriol. https://doi.org/10.1128/jb.00608-16 PubMedCentralCrossRefPubMedGoogle Scholar
- 36.Papadimitriou K, Alegria A, Bron PA, de Angelis M, Gobbetti M, Kleerebezem M, Lemos JA, Linares DM, Ross P, Stanton C, Turroni F, van Sinderen D, Varmanen P, Ventura M, Zuniga M, Tsakalidou E, Kok J (2016) Stress physiology of lactic acid bacteria. Microbiol Mol Biol Rev 80:837–890. https://doi.org/10.1128/mmbr.00076-15 CrossRefPubMedPubMedCentralGoogle Scholar
- 37.Papagianni M, Avramidis N (2011) Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation. Enzyme Microb Technol 49:197–202. https://doi.org/10.1016/j.enzmictec.2011.05.002 CrossRefPubMedGoogle Scholar
- 49.van de Guchte M, Penaud S, Grimaldi C, Barbe V, Bryson K, Nicolas P, Robert C, Oztas S, Mangenot S, Couloux A, Loux V, Dervyn R, Bossy R, Bolotin A, Batto JM, Walunas T, Gibrat JF, Bessieres P, Weissenbach J, Ehrlich SD, Maguin E (2006) The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci USA 103:9274–9279. https://doi.org/10.1073/pnas.0603024103 CrossRefPubMedGoogle Scholar
- 57.Zhai Z, Douillard FP, An H, Wang G, Guo X, Luo Y, Hao Y (2014) Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp bulgaricus CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb0677. Environ Microbiol 16:1524–1537. https://doi.org/10.1111/1462-2920.12280 CrossRefPubMedGoogle Scholar