title: Harnessing Candida tenuis and Pichia stipitis in whole-cell bioreductions of o-chloroacetophenone: stereoselectivity, cell activity, in situ substrate supply and product removal.
abstract: Generally, recombinant and native microorganisms can be employed as whole-cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H-dependent reduction of o-chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co-expressing xylose reductase and a dehydrogenase for co-enzyme recycling. Xylose-grown cells of C. tenuis and P. stipitis displayed specific o-chloroacetophenone reductase activities of 366 and 90 U gCDW (-1) , respectively, in the cell-free extracts. Fresh biomass was employed in batch reductions of 100 mM o-chloroacetophenone using glucose as co-substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a (S)-1-(2-chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 gCDW L(-1) of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7-fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo-like kinase 1 inhibitors).
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology TUG, A-8010 Graz, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Gruber
authors.forename: Stefan
authors.initials: S
authors.lastname: Krahulec
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
authors.forename: Regina
authors.initials: R
authors.lastname: Kratzer
mesh_terms: D000449:Aldehyde Reductase / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology*; D001709:Biotechnology / Q000379:methods*; D002175:Candida / Q000378:metabolism*; D005947:Glucose / Q000032:analysis / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D014994:Xylose / Q000032:analysis / Q000378:metabolism; D002721:omega-Chloroacetophenone / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009930:Organic Chemicals; D012997:Solvents; D009243:NAD; D002721:omega-Chloroacetophenone; D014994:Xylose; D000449:Aldehyde Reductase; D005947:Glucose