Skip to main content
SpringerLink
Log in

Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone

  • Applied Microbial and Cell Physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.

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

Similar content being viewed by others

References

  • Aguilera A (1986) Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae. Mol Gen Genet 204:310–316

    Article  CAS  PubMed  Google Scholar 

  • Almqvist F (1996) Synthesis of optically pure active bicyclo[2.2.2]octane derivatives—A study towards novel taxol mimetics and development of new chiral ligands for asymmetric catalysis. Doctoral thesis, Lund University

  • Almqvist F, Eklund L, Frejd T (1993) An improved procedure for the synthesis of bicyclo[2.2.2]octane-2,6-dione. Synth Commun 6:957–960

    Google Scholar 

  • Almqvist F, Torstensson L, Gudmunsson A, Frejd T (1997) New ligands for the titanium IV-induced catalytic asymmetric reduction of ketones with catecholborane. Angew Chem Int Engl 4:376–377

    Article  Google Scholar 

  • Botes A, Harvig D, van Dyk M, Sarvary I, Frejd T, Katz M, Hahn-Hägerdal B, Gorwa-Grauslund MF (2002) Screening of yeast species for the stereo-selective reduction of bicyclo[2.2.2]octane-2,6-dione. J Chem Soc Perkin Trans 1:1111–1114

    Article  Google Scholar 

  • Chartrain M, Randolph G, Moore J, Reider P, Robinson D, Buckland B (2001) Asymmetric bioreduction: application to the synthesis of pharmaceuticals. J Mol Cat B: Enzym 11:503–512

    Article  CAS  Google Scholar 

  • Chin-Joe I, Nelisse PM, Straathof AJ, Jongejan JA, Pronk JT, Heijnen JJ (2000) Hydrolytic activity in baker’s yeast limits the yield of asymmetric 3-oxo ester reduction. Biotechnol Bioeng 69:370–376

    Article  CAS  PubMed  Google Scholar 

  • Csuk R, Glänzer BI (1991) Baker’s yeast mediated transformations in organic chemistry. Chem Rev 91:49–97

    Article  CAS  Google Scholar 

  • D’Arrigo P, Pedrocchi-Fantoni G, Servi S (1997) Old and new synthetic capacities of baker’s yeast. Adv Appl Microbiol 44:81–123

    Article  PubMed  Google Scholar 

  • de Souza Pereira R (1998) The use of baker’s yeast in the generation of asymmetric centers to produce chiral drugs and other compounds. Crit Rev Biotechnol 18:25–83

    Article  Google Scholar 

  • Han K, Levenspiel O (1988) Extended monod kinetics for substrate, product, and cell inhibition. Biotechnol Bioeng 32:430–437

    Article  CAS  PubMed  Google Scholar 

  • Hilterhaus L, Liese A (2007) Building blocks. Adv Biochem Eng Biotechnol 105:133–173

    CAS  Google Scholar 

  • Jayasinghe LY, Kodituwakku D, Smallridge AJ, Trewhella MA (1994) The use of organic solvent systems in the yeast mediated reduction of ethyl acetoacetate. Bull Chem Soc Jpn 67:2528–2531

    Article  CAS  Google Scholar 

  • Johanson T, Katz M, Gorwa-Grauslund MF (2005) Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases. FEMS Yeast Res 5:513–525

    Article  CAS  PubMed  Google Scholar 

  • Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF (2005) Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 22:359–368

    CAS  PubMed  Google Scholar 

  • Katz M, Sarvary I, Frejd T, Hahn-Hägerdal B, Gorwa-Grauslund MF (2002) An improved stereoselective reduction of a bicyclic diketone by Saccharomyces cerevisiae combining process optimization and strain engineering. Appl Microbiol Biotechnol 59:641–648

    Article  CAS  PubMed  Google Scholar 

  • Katz M, Frejd T, Hahn-Hägerdal B, Gorwa-Grauslund MF (2003a) Efficient anaerobic whole cell stereoselective bioreduction with recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 84:573–582

    Article  CAS  PubMed  Google Scholar 

  • Katz M, Hahn-Hägerdal B, Gorwa-Grauslund MF (2003b) Screening of two complementary collections of Saccharomyces cerevisiae to identify enzymes involved in stereo-selective reduction of specific carbonyl compounds: an alternative to protein purification. Enz Microb Technol 33:163–172

    Article  CAS  Google Scholar 

  • Liese A (2002) Replacing chemical steps by biotransformations: industrial application and processes using biocatalysis. In: Drauz K, Waldmann H (eds.) Enzyme catalysis in organic synthesis. Wiley-VCH Verlag GmbH, Weinheim, pp. 1419–1459

    Chapter  Google Scholar 

  • Martin SF, White JB, Wagner R (1982) Alkoxide-accelerated sigmatropic rearrangements. A novel entry to the bicyclo[5.3.1.]undec-7-ene system of the taxane diterpenes. J Org Chem 47:3190–3192

    Article  CAS  Google Scholar 

  • Mori K, Nagano E (1990) Preparative bioorganic chemistry part 10. Asymmetric reduction of Bicyclo[2.2.2]octane-2,6-diones with baker’s yeast. Biocatalysis 3:25–36

    Article  CAS  Google Scholar 

  • Patel RN (2006) Biocatalysis: synthesis of chiral intermediates for drugs. Curr Opin Drug Discov Devel 9:741–764

    CAS  PubMed  Google Scholar 

  • Sarvary I, Almqvist F, Frejd T (2001) Asymmetric reduction of ketones with catecholborane using 2,6-BODOL complexes of titanium(IV) as catalysts. Chem Eur J 10:2158–2166

    Article  Google Scholar 

  • Sarvary I, Yiqian W, Frejd T (2002) Novel, cyclic and bicyclic 1,3-diols as catalysts for the diethylzinc addition to aldehydes. J Chem Soc Perkin Trans 15:645–651

    Article  Google Scholar 

  • Schmid A, Dordick JS, Kiener A, Wubbolts M, Witholt B (2001) Industrial biocatalysis today and tomorrow. Nature 409:258–268

    Article  CAS  PubMed  Google Scholar 

  • Servi S (1990) Baker’s yeast as a reagent in organic synthesis. Synthesis 1–25

  • Stewart JD (2000) Organic transformations catalyzed by engineered yeast cells and related systems. Curr Opin Biotechnol 11:363–368

    Article  CAS  PubMed  Google Scholar 

  • Widegren M, Dietz M, Friberg A, Frejd T, Hahn-Hägerdal B, Gorwa-Grauslund MF, Katz M (2006) The synthesis of bicyclo[2.2.2]octan-2,6-dione revisited. Synthesis Stuttgart 20:3527–3530

    Google Scholar 

Download references

Acknowledgement

We would like to thank Dr. Ed Van Niel for help with the Han-Levenspiel kinetic model, Nora Bieler for her assistance with the bioreductions and Prof. Bärbel Hahn-Hägerdal for critically reading the manuscript.

Author information

Author notes

  1. Andreas Rudolf

    Present address: Biosystems Department, Risø National Laboratory, Technical University of Denmark—DTU, P.O. Box 49, DK-4000, Roskilde, Denmark

Authors and Affiliations

  1. Department of Applied Microbiology, Lund University, P.O. Box 124, SE-22100, Lund, Sweden

    Ted Johanson, Magnus Carlquist & Marie F. Gorwa-Grauslund

  2. Department of Organic Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden

    Cecilia Olsson & Torbjörn Frejd

  3. Department of Chemical Engineering, Lund University, P.O. Box 124, SE-22100, Lund, Sweden

    Andreas Rudolf

Authors
  1. Ted Johanson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Magnus Carlquist
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cecilia Olsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andreas Rudolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Torbjörn Frejd
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marie F. Gorwa-Grauslund
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marie F. Gorwa-Grauslund.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Johanson, T., Carlquist, M., Olsson, C. et al. Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone. Appl Microbiol Biotechnol 77, 1111–1118 (2008). https://doi.org/10.1007/s00253-007-1240-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-007-1240-1

Keywords

Search

Navigation