Skip to main content
SpringerLink
Log in

Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereocomplex PLA with improved properties. In this study, we developed Saccharomyces cerevisiae strains for efficient production of D-LA. D-LA production was achieved by expressing highly stereospecific D-lactate dehydrogenase gene (ldhA, LEUM_1756) from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 in S. cerevisiae lacking natural LA production activity. D-LA consumption after glucose depletion was inhibited by deleting DLD1 encoding D-lactate dehydrogenase and JEN1 encoding monocarboxylate transporter. In addition, ethanol production was reduced by deleting PDC1 and ADH1 genes encoding major pyruvate decarboxylase and alcohol dehydrogenase, respectively, and glycerol production was eliminated by deleting GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase. LA tolerance of the engineered D-LA-producing strain was enhanced by adaptive evolution and overexpression of HAA1 encoding a transcriptional activator involved in weak acid stress response, resulting in effective D-LA production up to 48.9 g/L without neutralization. In a flask fed-batch fermentation under neutralizing condition, our evolved strain produced 112.0 g/L D-LA with a yield of 0.80 g/g glucose and a productivity of 2.2 g/(L · h).

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
Fig. 6

Similar content being viewed by others

References

  • Abbott DA, Suir E, van Maris AJ, Pronk JT (2008) Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae. Appl Environ Microbiol 74(18):5759–5768

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Abdel-Rahman MA, Tashiro Y, Sonomoto K (2013) Recent advances in lactic acid production by microbial fermentation processes. Biotechnol Adv 31(6):877–902

    Article  CAS  PubMed  Google Scholar 

  • Branduardi P, Sauer M, De Gioia L, Zampella G, Valli M, Mattanovich D, Porro D (2006) Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export. Microb Cell Fact 5(1):1–12

    Article  Google Scholar 

  • Cho BR, Lee P, Hahn JS (2014) CK2-dependent inhibitory phosphorylation is relieved by Ppt1 phosphatase for the ethanol stress-specific activation of Hsf1 in Saccharomyces cerevisiae. Mol Microbiol 93(2):306–316

    Article  CAS  PubMed  Google Scholar 

  • Collart MA, Oliviero S (2001) Preparation of yeast RNA. Curr Protoc Mol Biol 13(13):12

    PubMed  Google Scholar 

  • Colombié S, Dequin S, Sablayrolles JM (2003) Control of lactate production by Saccharomyces cerevisiae expressing a bacterial LDH gene. Enzyme Microb Technol 33(1):38–46

    Article  Google Scholar 

  • Dato L, Berterame N, Ricci M, Paganoni P, Palmieri L, Porro D, Branduardi P (2014) Changes in SAM2 expression affect lactic acid tolerance and lactic acid production in Saccharomyces cerevisiae. Microb Cell Factories 13:147

    Google Scholar 

  • Garlotta D (2001) A literature review of poly(lactic acid). J Polym Environ 9(2):63–84

    Article  CAS  Google Scholar 

  • Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30(6), e23

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hofvendahl K, Hahn-Hägerdal B (2000) Factors affecting the fermentative lactic acid production from renewable resources1. Enzyme Microb Technol 26(2–4):87–107

    Article  CAS  PubMed  Google Scholar 

  • Ida Y, Hirasawa T, Furusawa C, Shimizu H (2013) Utilization of Saccharomyces cerevisiae recombinant strain incapable of both ethanol and glycerol biosynthesis for anaerobic bioproduction. Appl Microbiol Biotechnol 97(11):4811–4819

    Article  CAS  PubMed  Google Scholar 

  • Ikada Y, Jamshidi K, Tsuji H, Hyon SH (1987) Stereocomplex formation between enantiomeric poly(lactides). Macromolecules 20(4):904–906

    Article  CAS  Google Scholar 

  • Inaba T, Watanabe D, Yoshiyama Y, Tanaka K, Ogawa J, Takagi H, Shimoi H, Shima J (2013) An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses. AMB express 3(1):74

    Article  PubMed Central  PubMed  Google Scholar 

  • Ishida N, Saitoh S, Tokuhiro K, Nagamori E, Matsuyama T, Kitamoto K, Takahashi H (2005) Efficient production of L-lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene. Appl Environ Microbiol 71(4):1964–1970

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ishida N, Saitoh S, Onishi T, Tokuhiro K, Nagamori E, Kitamoto K, Takahashi H (2006a) The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production. Biosci, Biotechnol, Biochem 70(5):1148–1153

    Article  CAS  Google Scholar 

  • Ishida N, Suzuki T, Tokuhiro K, Nagamori E, Onishi T, Saitoh S, Kitamoto K, Takahashi H (2006b) D-lactic acid production by metabolically engineered Saccharomyces cerevisiae. J Biosci Bioeng 101(2):172–177

    Article  CAS  PubMed  Google Scholar 

  • Jun C, Sa YS, Gu S-A, Joo JC, Kim S, Kim K-J, Kim YH (2013) Discovery and characterization of a thermostable D-lactate dehydrogenase from Lactobacillus jensenii through genome mining. Process Biochem 48(1):109–117

    Article  CAS  Google Scholar 

  • Kawahata M, Masaki K, Fujii T, Iefuji H (2006) Yeast genes involved in response to lactic acid and acetic acid: acidic conditions caused by the organic acids in Saccharomyces cerevisiae cultures induce expression of intracellular metal metabolism genes regulated by Aft1p. FEMS Yeast Res 6(6):924–936

    Article  CAS  PubMed  Google Scholar 

  • Kim S, Hahn J-S (2015) Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing. Metab Eng 31:94–101

    Article  CAS  PubMed  Google Scholar 

  • Lee JY, Kang CD, Lee SH, Park YK, Cho KM (2015) Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L-lactic acid. Biotechnol Bioeng 112(4):751–758

    Article  CAS  PubMed  Google Scholar 

  • Li L, Eom H-J, Park J-M, Seo E, Ahn JE, Kim T-J, Kim JH, Han NS (2012) Characterization of the major dehydrogenase related to D-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293. Enzyme Microb Technol 51(5):274–279

    Article  PubMed  Google Scholar 

  • Lodi T, Alberti A, Guiard B, Ferrero I (1999) Regulation of the Saccharomyces cerevisiae DLD1 gene encoding the mitochondrial protein D-lactate ferricytochrome c oxidoreductase by HAP1 and HAP2/3/4/5. Mol Gen Genet 262(4–5):623–632

    Article  CAS  PubMed  Google Scholar 

  • Lodi T, Fontanesi F, Guiard B (2002) Co-ordinate regulation of lactate metabolism genes in yeast: the role of the lactate permease gene JEN1. Mol Genet Genomics 266(5):838–847

    Article  CAS  PubMed  Google Scholar 

  • Mira NP, Teixeira MC, Sá-Correia I (2010) Adaptive response and tolerance to weak acids in Saccharomyces cerevisiae: a genome-wide view. OMICS: J Integrative Biol 14(5):525–540

    Article  CAS  Google Scholar 

  • Mira NP, Henriques SF, Keller G, Teixeira MC, Matos RG, Arraiano CM, Winge DR, Sa-Correia I (2011) Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress. Nucleic Acids Res 39(16):6896–907

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156(1):119–122

    Article  CAS  PubMed  Google Scholar 

  • Okino S, Suda M, Fujikura K, Inui M, Yukawa H (2008) Production of D-lactic acid by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 78(3):449–454

    Article  CAS  PubMed  Google Scholar 

  • Pacheco A, Talaia G, Sá-Pessoa J, Bessa D, Gonçalves MJ, Moreira R, Paiva S, Casal M, Queirós O (2012) Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2. FEMS Yeast Res 12(3):375–381

    Article  CAS  PubMed  Google Scholar 

  • Porro D, Brambilla L, Ranzi BM, Martegani E, Alberghina L (1995) Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid. Biotechnol Prog 11(3):294–298

    Article  CAS  PubMed  Google Scholar 

  • Saitoh S, Ishida N, Onishi T, Tokuhiro K, Nagamori E, Kitamoto K, Takahashi H (2005) Genetically engineered wine yeast produces a high concentration of L-lactic acid of extremely high optical purity. Appl Environ Microbiol 71(5):2789–2792

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122(1):19–27

    PubMed Central  CAS  PubMed  Google Scholar 

  • Skory C (2003) Lactic acid production by Saccharomyces cerevisiae expressing a Rhizopus oryzae lactate dehydrogenase gene. J Ind Microbiol Biotechnol 30(1):22–27

    Article  CAS  PubMed  Google Scholar 

  • Sugiyama M, Akase S-P, Nakanishi R, Horie H, Kaneko Y, Harashima S (2014) Nuclear localization of Haa1, which is linked to its phosphorylation status, mediates lactic acid tolerance in Saccharomyces cerevisiae. Appl Environ Microbiol 80(11):3488–3495

    Article  PubMed Central  PubMed  Google Scholar 

  • Suzuki T, Sakamoto T, Sugiyama M, Ishida N, Kambe H, Obata S, Kaneko Y, Takahashi H, Harashima S (2013) Disruption of multiple genes whose deletion causes lactic-acid resistance improves lactic-acid resistance and productivity in Saccharomyces cerevisiae. J Biosci Bioeng 115(5):467–474

    Article  CAS  PubMed  Google Scholar 

  • Tokuhiro K, Ishida N, Nagamori E, Saitoh S, Onishi T, Kondo A, Takahashi H (2009) Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene. Appl Microbiol Biotechnol 82(5):883–890

    Article  CAS  PubMed  Google Scholar 

  • Vaidya AN, Pandey RA, Mudliar S, Kumar MS, Chakrabarti T, Devotta S (2005) Production and recovery of lactic acid for polylactide—an overview. Crit Rev Environ Sci Technol 35(5):429–467

    Article  CAS  Google Scholar 

  • Valli M, Sauer M, Branduardi P, Borth N, Porro D, Mattanovich D (2006) Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH. Appl Environ Microbiol 72(8):5492–5499

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • van Maris AJA, Geertman J-MA, Vermeulen A, Groothuizen MK, Winkler AA, Piper MDW, van Dijken JP, Pronk JT (2004a) Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Appl Environ Microbiol 70(1):159–166

    Article  PubMed Central  PubMed  Google Scholar 

  • van Maris AJA, Winkler AA, Porro D, van Dijken JP, Pronk JT (2004b) Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export. Appl Environ Microbiol 70(5):2898–2905

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. N. S. Han for providing a plasmid.

Author information

Authors and Affiliations

  1. School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea

    Seung-Ho Baek, Eunice Y. Kwon & Ji-Sook Hahn

  2. Department of Chemical Engineering, Kwangwoon University, 20 Gwangun-ro, Nowon-gu, Seoul, 01897, Republic of Korea

    Yong Hwan Kim

Authors
  1. Seung-Ho Baek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eunice Y. Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yong Hwan Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ji-Sook Hahn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ji-Sook Hahn.

Ethics declarations

Funding

This study was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (NRF-2015R1A2A2A01005429).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Baek, SH., Kwon, E.Y., Kim, Y.H. et al. Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae . Appl Microbiol Biotechnol 100, 2737–2748 (2016). https://doi.org/10.1007/s00253-015-7174-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-7174-0

Keywords

Search

Navigation