Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Investigating the effectiveness of DNA microarray analysis for identifying the genes involved in l-lactate production by Saccharomyces cerevisiae

  • 218 Accesses

  • 13 Citations

Abstract

In order to determine whether transcriptome data obtained by DNA microarray analysis could be used to identify the genes involved in target metabolite production, we tried to identify the genes involved in l-lactate production by l-lactate-producing recombinant Saccharomyces cerevisiae strains. We obtained DNA microarray data for these strains. Plasmids carrying lactic acid bacteria, bovine, and human l-lactate dehydrogenase (LDH) genes were introduced into PDC1-disrupted S. cerevisiae strains. l-Lactate productivity of the strains harboring the human and bovine LDH genes was higher than that of the strains harboring lactic acid bacteria LDH genes. DNA microarray analysis revealed that the expression of 388 genes was significantly altered in the strains with the human and bovine LDH genes. Of these, the l-lactate productivity of human LDH-harboring deletion strains of 289 genes was compared with that of the standard and 56 randomly selected deletion strains containing the same LDH gene to validate the effectiveness of DNA microarray analysis for identifying the genes responsible for l-lactate production in the recombinant strains. Only deletion strains of the genes selected on the basis of the DNA microarray data showed significantly altered l-lactate production as compared to the standard and the randomly selected deletion strains. Our results indicated that the genes related to l-lactate production could be successfully identified by selecting the genes that exhibited significantly altered expression on DNA microarray analysis, and the effectiveness of DNA microarray analysis for identifying the genes responsible for l-lactate production was discussed.

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

Fig. 1
Fig. 2
Fig. 3

References

  1. 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 74:5759–5768

  2. Adachi E, Torigoe M, Sugiyama M, Nikawa J, Shimizu K (1998) Modification of metabolic pathways of Saccharomyces cerevisiae by the expression of lactate dehydrogenase and deletion of pyruvate decarboxylase genes for the lactic acid fermentation at low pH value. J Ferment Bioeng 86:284–289

  3. Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G (2004) GO::TermFinder-open source software for accessing gene ontology information and finding significantly enriched gene ontology terms associated with a list of genes. Bioinformatics 20:3710–3715

  4. Dien BS, Nichols NN, Bothast RJ (2001) Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars. J Ind Microbiol Biotech 27:259–264

  5. Harris DM, Van Der Krogt ZA, Klaassen P, Raamsdonk LM, Hage S, Van Den Berg MA, Bovenberg RA, Pronk JT, Daran J-M (2009) Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production. BMC Genomics 10:75

  6. Hirasawa T, Nakakura Y, Yoshikawa K, Ashitani K, Nagahisa K, Furusawa C, Katakura Y, Shimizu H, Shioya S (2006) Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray. Appl Microbiol Biotechnol 70:346–357

  7. Hirasawa T, Yoshikawa K, Nakakura Y, Nagahisa K, Furusawa C, Katakura Y, Shimizu H, Shioya S (2007) Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis. J Biotechnol 131:34–44

  8. Imaizumi A, Takikawa R, Koseki C, Usuda Y, Yasueda H, Kojima H, Matsui K, Sugimoto S (2005) Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli. J Biotechnol 117:111–118

  9. 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 71:1964–1970

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

  11. Kobayashi M, Nagahisa K, Shimizu H, Shioya S (2006) Simultaneous control of apparent extract and volatile compounds concentrations in low-malt beer fermentation. Appl Microbiol Biotechnol 73:549–558

  12. Köhrer K, Domdey H (1991) Preparation of high molecular weight RNA. Methods Enzymol 194:398–405

  13. Lum AM, Huang J, Hutchinson CR, Kao CM (2004) Reverse engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays. Metab Eng 6:186–196

  14. Nagino K, Nomura O, Takii Y, Myomoto A, Ichikawa M, Nakamura F, Higasa M, Akiyama H, Nobumasa H, Shiojima S, Tsujimoto G (2006) Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification. J Biochem 139:697–703

  15. Ninomiya-Tsuji J, Nomoto S, Yasuda H, Reed SI, Matsumoto K (1991) Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation. Proc Natl Acad Sci U S A 88:9006–9010

  16. Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K, Ikeda M (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl Microbiol Biotechnol 58:217–223

  17. Ookubo A, Hirasawa T, Yoshikawa K, Nagahisa K, Furusawa C, Shimizu H (2008) Improvement of L-lactate production by CYB2 gene disruption in a recombinant Saccharomyces cerevisiae strain under low pH condition. Biosci Biotechnol Biochem 72:3063–3066

  18. 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 11:294–298

  19. 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 71:2789–2792

  20. Shibuya H, Irie K, Ninomiya-Tsuji J, Goebl M, Taniguchi T, Matsumoto K (1992) New human gene encoding a positive modulator of HIV Tat-mediated transactivation. Nature 357:700–702

  21. Sindelar G, Wendisch VF (2007) Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes. Appl Microbiol Biotechnol 76:677–689

  22. 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 82:883–890

  23. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M'rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Véronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906

  24. Workman C, Jensen LJ, Jarmer H, Berka R, Gautier L, Nielser HB, Saxild HH, Nielsen C, Brunak S, Knudsen S (2002) A new non-linear normalization method for reducing variability in DNA microarray experiments. Genome Biol 3:research0048

  25. Yoshikawa K, Tanaka T, Furusawa C, Nagahisa K, Hirasawa T, Shimizu H (2009) Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae. FEMS Yeast Res 9:32–44

  26. Zhou S, Causey TB, Hasona A, Shanmugam KT, Ingram LO (2003) Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl Environ Microbiol 69:399–407

  27. Zhu J, Shimizu K (2004) The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Appl Microbiol Biotechnol 64:367–375

Download references

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research (B) to HS (21360401) from Japan Society for Promotion of Science and Grants-in-Aid for Young Scientists (B) to TH (21780071) and CF (20700270) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported in part by the Global COE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Author information

Correspondence to Hiroshi Shimizu.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

Table S1

Gene expression profiles in strain Nos. 48 and 49 in comparison with that in No. 11 (PDF 487 kb)

Table S2

l-lactate production (g/l) in the pTRS48-transformed disruptants of the genes whose expression was upregulated in both Nos. 48 and 49 strains (PDF 46 kb)

Table S3

l-lactate production in the pTRS48-transformed disruptants of the genes whose expression was downregulated in both Nos. 48 and 49 strains (PDF 44 kb)

Table S4

l-lactate production in the pTRS48-transformed disruptants of the genes selected randomly (PDF 37 kb)

Table S5

l-lactate production (g/l) in the standard strain (BY4742 HIS3::kanMX4) (PDF 36 kb)

Supplemental materials

(PDF 187 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hirasawa, T., Ookubo, A., Yoshikawa, K. et al. Investigating the effectiveness of DNA microarray analysis for identifying the genes involved in l-lactate production by Saccharomyces cerevisiae . Appl Microbiol Biotechnol 84, 1149–1159 (2009). https://doi.org/10.1007/s00253-009-2209-z

Download citation

Keywords

  • Saccharomyces cerevisiae
  • l-Lactate
  • DNA microarray
  • Knockout strain library