Efficient Pathways to High-Value Bioactives
ID: 3930

Abstract: Won Jae Choi

Chiral epoxides are high value-added intermediates for the synthesis of pharmaceuticals, agrochemicals, as well as versatile fine chemicals and have broad scope of market demand for their applications [1]. A major challenge in modern organic chemistry is to generate such compounds in high yields, with high stereo- and regioselectivities. Epoxide hydrolases (EH) have shown promise as biocatalysts for the preparation of chiral epoxides and vicinal diols. They exhibit high enantioselectivities for their substrates, and can be effectively used in the resolution of racemic epoxides through enantioselective hydrolysis [2]. Selective hydrolysis of racemic epoxide can generate both corresponding diol and unreacted remaining epoxide with high enantiomeric excess (ee) value. The clear potential demonstrated by the microbial EHs has prompted researchers to explore their use in the synthesis of epoxides and diols with high ee values [3]. Microbial kinetic resolutions of racemic epoxides were attempted for the production of chiral epoxides via EH-mediated enantioselective hydrolysis. (S)-Ethyl 3,4-epoxybutyrate with ee higher than 99% was obtained from its racemate with a yield of 46 % (theoretically 50% maximum) by whole cells of newly isolated Acinetobacter baumannii containing EH.

References

[1] A. Archelas and R. Furstoss, Curr. Opin. Chem. Biol. 5, 112 (2001).

[2] A. Steinreiber and K. Faber, Curr. Opin. Biotechnol. 12, 552 (2001).

[3] W. J. Choi and C. Y. Choi, Biotechnol. Bioprocess Eng. 10, 167 (2005).



Paul Dalby

Edward G. Hibbert, Mark E. B. Smith, Mathew P. Martin, Kirsty Smithies, Ursula Kaulmann, Jenny A. Littlechild, Helen C. Hailes, John M. Ward and Paul A. Dalby*

Department of Biochemical Engineering, University College London, UK; Department of Chemistry, University College London, UK; Department of Biochemistry and Molecular Biology, University College London, UK

E-mail: p.dalby@ucl.ac.uk

The aminodiol functionality is present in many natural and synthetic biologically active molecules including antibiotics[1], alkaloids[2] and amino sugars[3]. Though methodology exists for the asymmetric synthesis of aminoalcohols and aminodiols, such as via the Sharpless aminohydroxylation reaction[4], the methods are generally step intensive and/or consume expensive and sometimes toxic catalysts or chiral auxilliaries[5].

An elegant de novo biocatalytic pathway has enabled the synthesis of chiral aminodiols[6] which is now being enhanced to an industrially useful scale. A multi-disciplinary team is working towards the engineering and directed evolution of novel transketolase (TK) mutants and transaminases (TAm) capable of processing a multitude of aldehyde substrates into chiral aminodiols via two sequential biotransformations. The group is also comparing the relative efficiencies of making aminodiols using biocatalytic approaches and chemical methods.

The design of targeted mutant libraries based upon both phylogenetic and structural information, and their characterisation against a range of substrates including cyclic aldehydes, will be described. Novel transketolase mutants obtained will be described, together with recent structural insights into their altered substrate binding and enantioselectivities.

[1] G. Wu, D. P. Schumacher, W. Tormos, J. E. Clark and B. L. Murphy, J. Org. Chem., 1997, 62, 2996.

[2] M. T. Reetz, Angew. Chem., Int. Ed. Engl., 1991, 30, 1531.

[3] A. B. Hughes and A. J. Rudge, Nat. Prod. Rep., 1994, 11, 135.

[4] K. B. Sharpless and G. Li, Angew. Chem., Int. Ed. Engl., 1996, 108, 449.

[5] D. A. Evans and A. E. Weber, J. Am. Chem. Soc., 1987, 109, 7151.

[6] C. U. Ingram, M. Bommer, M. E. B. Smith, P. A. Dalby, J. M. Ward, H. C. Hailes, and G. J. Lye, Biotech. Bioeng., 2007, 96, 559-569.

[7] E.G. Hibbert, T. Senussi, S. J.Costelloe, W. Lei, M. E. B. Smith, J. M. Ward, H. C. Hailes, and P. A. Dalby, J. Biotechnol., 2007, 131, 425-432.

[8] M. E. B. Smith, E.G. Hibbert, A.B. Jones, P. A. Dalby and H. C. Hailes, Adv. Syn. Catal., 2008, 350, 2631-2638.



Jens Schrader

Plant terpenoids play an important role as bioactive molecules in nature and many of these compounds are of commercial interest, e.g. as flavors, fragrances, cosmetic or pharmaceutic ingredients. Regio- and stereoselective biooxidation of terpenoid precursors, which are abundantly available from Nature, represents a powerful alternative to chemical synthesis; however, toxic effects towards microbial cells and low water solubility are inherent properties of terpenoids making biocatalytic oxyfunctionalization a real challenge [1] which should be addressed by both genetic and bioprocess engineering. This contribution will describe three recent advances of our current bioprocess development efforts covering wildtype and recombinant microorganisms as well as isolated enzymes as biocatalysts:

1. The biotransformation of the monocyclic monoterpene (+)-limonene, a cheap by-product of the citrus processing industry, to (+)-perillic acid with a solvent tolerant Pseudomonas putida strain was dramatically improved to > 30 g L-1, the highest product concentration reported so far for a microbial monoterpene oxidation. The monoterpenoic acid is being discussed as a promising agent for different applications, e.g. for natural preservation in cosmetics and for anti-cancer treatment. Key to success was the integration of an in-situ product recovery (ISPR) to alleviate product inhibition [2].

2. The oxyfunctionalization of the bicyclic monoterpene a-pinene to valuable flavor compounds or precursors thereof was performed with E. coli whole cells overexpressing a quintuple mutant of a P450 monooxygenase from Bacillus megaterium (P450BM-3). To compensate for the increased NADPH demand a heterologous cofactor regeneration system comprising a glucose facilitator and a glucose dehydrogenase was coexpressed and an aqueous-organic two-liquid-phase bioprocess for in-situ precursor delivery and product recovery was developed yielding > 1 g Laq-1 total product for the first time [3].

3. The recently discovered enzyme family of carotenoid cleavage dioxygenases (CCD) opens a direct access to valuable bioactives, such as flavors, fragrances, vitamins and colorants from tetraterpenoids (carotenoids) by specific oxidative cleavage of C-C double bonds. AtCCD1 from Arabidopsis thaliana cleaves different carotenoids symmetrically at the 9-10 / 9'-10' positions yielding C13 norisoprenoids, such as the natural flavor compound ß-ionone. We addressed the main hurdle for an in vitro application of CCD, the poor substrate solubility, by medium engineering based on micellar reaction systems [4].

[1] Schrader (2007) In: Berger, R.G. (ed) Flavours and Fragrances, Springer, Berlin, p. 507-574.

[2] Mirata et al. (2008) in prep.

[3] Schewe et al. (2008) Appl Microbiol Biotechnol 78: 55-65; Schewe et al (2008) Appl Microbiol Biotechnol, in prep.

[4] Schilling et al. (2007) Appl Microbiol Biotechnol 75, 829-836; Schilling et al. (2008) Biotech Lett 30, 701-706







Moderator: Paul Dalby, UCL Biomedica PLC (United Kingdom)

Presenter 1: Biocatalytic Production of Chiral Epoxides
Won Jae Choi, Institute of chemical and engineering sciences, (Singapore)  [Confirmed]

Presenter 2: De Novo Pathway Design and Engineering of Enzyme Specificity and Enantioselectivity for Improved Ketodiol and Aminodiol Synthesis 
Paul Dalby, UCL Biomedica PLC, (United Kingdom)  [Confirmed]

Presenter 3: Biooxidation of Terpenoids for the Synthesis of Valuable Bioactives 
Jens Schrader, DECHEMA E.V., (Germany)  [Confirmed]

Presenter 4 (if necessary): {Presenter4PresentationTitle} 
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Panel Organizer:
Matthew Carr, Biotechnology Industry Organization, (United States)

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