Skip to main content
SpringerLink
Log in

Cloning and characterization of a l-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36–38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Agler MT, Spirito CM, Usack JG, Werner JJ, Angenent LT (2012) Chain elongation with reactor microbiomes: upgrading dilute ethanol to medium-chain carboxylates. Energy Environ Sci 5:8189–8192

    CAS  Google Scholar 

  • Andersen SJ, De Groof V, Khor WC, Roume H, Props R, Coma M, Rabaey K (2017) A Clostridium Group IV species dominates and suppresses a mixed culture fermentation by tolerance to medium chain fatty acids products. Front Bioeng Biotechnol 5:1–10

    Google Scholar 

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201

    CAS  PubMed  Google Scholar 

  • Cavalcante WDA, Leitão RC, Gehring TA, Angenent LT, Santaella ST (2017) Anaerobic fermentation for n-caproic acid production: a review. Process Biochem 54:106–119

    CAS  Google Scholar 

  • Chen WS, Ye Y, Steinbusch KJJ, Strik DPBTB, Buisman CJN (2016) Methanol as an alternative electron donor in chain elongation for butyrate and caproate formation. Biomass Bioenerg 93:201–208

    CAS  Google Scholar 

  • Collins SS, Keeton JT, Smith SB (1991) Lactate dehydrogenase enzyme activity in raw, cured, and heated porcine muscle. J Agric Food Chem 39:1294–1297

    CAS  Google Scholar 

  • Contag PR, Williams MG, Rogers P (1990) Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli. Appl Environ Microbiol 56:3760–3765

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cuff JA, Barton GJ (2000) Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40:502–511

    CAS  PubMed  Google Scholar 

  • Dietrich C, Nato A, Bost B, Le Marechal P, Guyonvarch A (2009) Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum. Microbiology 155:1360–1375

    CAS  PubMed  Google Scholar 

  • Dong H, Wang Y, Zhao Q, Han H, Zhu S, Li L, Wu Y, Huang B (2014) Molecular cloning and characterization of lactate dehydrogenase gene from Eimeria tenella. Parasitol Res 113:2915–2923

    PubMed  Google Scholar 

  • Evans JD, Martin SA (2002) Cloning of the L-lactate dehydrogenase gene from the ruminal bacterium Selenomonasruminantium HD4. Curr Microbiol 44:155–160

    CAS  PubMed  Google Scholar 

  • Fu Y, Sun X, Zhu H, Jiang R, Luo X, Yin L (2018) An optimized fed-batch culture strategy integrated with a one-step fermentation improves L-lactic acid production by Rhizopusoryzae. World J Microbiol Biotechnol 34:74–82

    PubMed  Google Scholar 

  • Gaspar P, Neves AR, Ramos A, Gasson MJ, Shearman CA, Santos H (2004) Engineering Lactococcuslactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system. Appl Environ Microbiol 70:1466–1474

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gokarn RR, Eiteman MA, Altman E (2000) Metabolic analysis of Escherichiacoli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase. Appl Environ Microbiol 66:1844–1850

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hahnke S, Abendroth C, Langer T, Codoner FM, Ramm P, Porcar M, Luschnig O, Klocke M (2018) Complete genome sequence of a new ruminococcaceae bacterium isolated from anaerobic biomass hydrolysis. Genome Announc 6:1–2

    Google Scholar 

  • Hannenhalli SS, Russell RB (2000) Analysis and prediction of functional sub-types from protein sequence alignments. J Mol Biol 303:61–76

    CAS  PubMed  Google Scholar 

  • Kim SG, Jang S, Lim JH, Jeon BS, Kim J, Kim KH, Sang BI, Jung GY (2018) Optimization of hexanoic acid production in recombinant Escherichiacoli by precise flux rebalancing. Bioresour Technol 247:1253–1257

    CAS  PubMed  Google Scholar 

  • Kouassi GK, Anantheswaran RC, Knabel SJ, Floros JD (2007) Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk. J Agric Food Chem 55:9520–9529

    CAS  PubMed  Google Scholar 

  • Kucek LA, Nguyen M, Angenent LT (2016) Conversion of L-lactate into n-caproate by a continuously fed reactor microbiome. Water Res 93:163–171

    CAS  PubMed  Google Scholar 

  • Lee JH, Choi MH, Park JY, Kang HK, Ryu HW, Sunwo CS, Wee YJ, Park KD, Kim DW, Kim D (2004) Cloning and characterization of the lactate dehydrogenase genes from Lactobacillus sp. RKY2. Biotechnol Bioprocess Eng 9:318–322

    CAS  Google Scholar 

  • Li X (2008) Purification and partial characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA). J Agric Food Chem 56:2392–2399

    CAS  PubMed  Google Scholar 

  • Louis P, Flint HJ (2009) Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett 294:1–8

    CAS  PubMed  Google Scholar 

  • Lu G, Hu X, Peng Z, Xie H, Li Y, Wu Z, Yu X (2006) Expression and characterization of lactate dehydrogenase from Schistosomajaponicum. Parasitol Res 99:593–596

    PubMed  Google Scholar 

  • Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Tasneem A, Thanki N, Yamashita RA, Zhang D, Zhang N, Bryant SH (2009) CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res 37:D205-210

    CAS  PubMed  Google Scholar 

  • Nadeem MS, Al-Ghamdi MA, Khan JA, Sadath S, Al-Malki A (2018) Recombinant production and biochemical and in silico characterization of lactate dehydrogenase from Geobacillus thermodenitrificans DSM-465. Electron J Biotechnol 35:18–24

    CAS  Google Scholar 

  • Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR (2001) Genome sequence and comparative analysis of the solvent-producing bacterium Clostridiumacetobutylicum. J Bacteriol 183:4823–4838

    CAS  PubMed  PubMed Central  Google Scholar 

  • Özkan M, Yllmaz EI, Lynd LR, Özcengiz G (2004) Cloning and expression of the Clostridiumthermocellum L-lactate dehydrogenase gene in Escherichiacoli and enzyme characterization1. Can J Microbiol 50:845–851

    PubMed  Google Scholar 

  • Ozkan M, Erhan E, Terzi O, Tan I, Ozoner SK (2009) Thermostable amperometric lactate biosensor with Clostridiumthermocellum L-LDH for the measurement of blood lactate. Talanta 79:1412–1417

    CAS  PubMed  Google Scholar 

  • Scott KP, Martin JC, Mrazek J, Flint HJ (2008) Transfer of conjugative elements from rumen and human Firmicutes bacteria to Roseburiainulinivorans. Appl Environ Microbiol 74:3915–3917

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sharkey MA, Maher MA, Guyonvarch A, Engel PC (2011) Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined. Arch Microbiol 193:731–740

    CAS  PubMed  Google Scholar 

  • Singh V, Kaushal DC, Rathaur S, Kumar N, Kaushal NA (2012) Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase. Protein Expr Purif 84:195–203

    CAS  PubMed  Google Scholar 

  • Singhvi M, Zendo T, Iida H, Gokhale D, Sonomoto K (2017) Stimulation of d- and l-lactate dehydrogenases transcriptional levels in presence of diammonium hydrogen phosphate resulting to enhanced lactic acid production by Lactobacillus strain. J Biosci Bioeng 124:674–679

    CAS  PubMed  Google Scholar 

  • Skory CD (2000) Isolation and expression of lactate dehydrogenase genes from Rhizopusoryzae. Appl Environ Microbiol 66:2343–2348

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stevenson DM, Weimer PJ (2005) Expression of 17 genes in Clostridiumthermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. Appl Environ Microbiol 71:4672–4678

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sun L, Zhang C, Lyu P, Wang Y, Wang L, Yu B (2016) Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacilluscoagulans. Sci Rep 6:37916–37923

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tao Y, Hu X, Zhu X, Jin H, Xu Z, Tang Q, Li X (2016) Production of butyrate from lactate by a newly isolated Clostridium sp. BPY5. Appl Biochem Biotechnol 179:361–374

    CAS  PubMed  Google Scholar 

  • Tao Y, Zhu X, Wang H, Wang Y, Li X, Jin H, Ru J (2017) Complete genome sequence of Ruminococcaceae bacterium CPB6: a newly isolated culture for efficient n-caproic acid production from lactate. J Biotechnol 259:91–94

    CAS  PubMed  Google Scholar 

  • Tebianian M, Hoseini AZ, Ebrahimi SM, Memarnejadian A, Mokarram AR, Mahdavi M, Sohrabi N, Taghizadeh M (2011) Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70. Biologicals 39:143–148

    CAS  PubMed  Google Scholar 

  • Toth J, Ismaiel AA, Chen JS (1999) The ald gene, encoding a coenzyme A-acylating aldehyde dehydrogenase, distinguishes Clostridiumbeijerinckii and two other solvent-producing Clostridia from Clostridiumacetobutylicum. Appl Environ Microbiol 65:4973–4980

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Y, Li X, Milne CB, Janssen H, Lin W, Phan G, Huiying Hu, Jin Y-S (2013) Development of a gene knockout system using mobile Group II Introns (Targetron) and genetic disruption of acid production pathways in Clostridiumbeijerinckii. Appl Environ Microbiol 79:5853–5863

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang H, Li X, Wang Y, Tao Y, Lu S, Zhu X, Li D (2018) Improvement of n-caproic acid production with Ruminococcaceae bacterium CPB6: selection of electron acceptors and carbon sources and optimization of the culture medium. Microb Cell Fact 17:99–108

    PubMed  PubMed Central  Google Scholar 

  • Wang P, Zhang J, Feng J, Wang S, Guo L, Wang Y, Lee YY, Taylor S, McDonald T, Wang Y (2019) Enhancement of acid re-assimilation and biosolvent production in Clostridiumsaccharoperbutylacetonicum through metabolic engineering for efficient biofuel production from lignocellulosic biomass. Bioresour Technol 281:217–225

    CAS  PubMed  Google Scholar 

  • Wu Q, Bao X, Guo W, Wang B, Li Y, Luo H, Wang H, Ren N (2019) Medium chain carboxylic acids production from waste biomass: current advances and perspectives. Biotechnol Adv 37:599–615

    CAS  PubMed  Google Scholar 

  • Xiao Y, Francke C, Abee T, Wells-Bennik MHJ (2011) Clostridial spore germination versus bacilli: genome mining and current insights. Food Microbiol 28:266–274

    PubMed  Google Scholar 

  • Zhang QL, Yang M, Zhao YY, Zhang SZ, He QH, Meng XY, Tan WR (2014) Cloning and characterization of lactate dehydrogenase C4 from Ochotonacurzoniae. Mol Biol 48:105–112

    CAS  Google Scholar 

  • Zhou Q, Shao WL (2010) Molecular genetic characterization of the thermostable L-lactate dehydrogenase gene (ldhL) of Thermoanaerobacterethanolicus JW200 and biochemical characterization of the enzyme. Biochemistry (Mosc) 75:526–530

    CAS  Google Scholar 

  • Zhou S, Shanmugam KT, Ingram LO (2003) Functional replacement of the Escherichiacoli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcusacidilactici. Appl Environ Microbiol 69:2237–2244

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu X, Tao Y, Liang C, Li X, Wei N, Zhang W, Zhou Y, Yang Y, Bo T (2015) The synthesis of n-caproate from lactate: a new efficient process for medium-chain carboxylates production. Sci Rep 5:14360–14369

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu X, Zhou Y, Wang Y, Wu T, Li X, Li D, Tao Y (2017) Production of high-concentration n-caproic acid from lactate through fermentation using a newly isolated Ruminococcaceae bacterium CPB6. Biotechnol Biofuels 10:102–114

    PubMed  PubMed Central  Google Scholar 

  • Zoraghi R, See RH, Gong H, Lian T, Swayze R, Finlay BB, Brunham RC, McMaster WR, Reiner NE (2010) Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcusaureus. Biochemistry 49:7733–7747

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the Natural Science Foundation of China (Grant No. 31770090), the Open-foundation project of CAS Key Laboratory of Environmental and Applied Microbiology (KLCAS-2017-01).

Author information

Author notes

  1. Qingzhuoma Yang and Cuicui Wei have contributed equally to the article.

Authors and Affiliations

  1. CAS Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, University of Chinese Academy of Sciences, Chengdu, 610041, China

    Qingzhuoma Yang, Cuicui Wei & Yong Tao

  2. BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China

    Shengtao Guo

  3. Faculty of Bioengineering, Sichuan University of Science & Engineering, Xueyuan Street 180#, Huixing Rd., Zigong, 643000, China

    Jun Liu & Yong Tao

Authors
  1. Qingzhuoma Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cuicui Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shengtao Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yong Tao.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2947 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Q., Wei, C., Guo, S. et al. Cloning and characterization of a l-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6. World J Microbiol Biotechnol 36, 182 (2020). https://doi.org/10.1007/s11274-020-02958-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11274-020-02958-4

Keywords

Search

Navigation