title: A highly productive, whole-cell DERA chemoenzymatic process for production of key lactonized side-chain intermediates in statin synthesis.
abstract: Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L(-1) h(-1) (up to 50 g L(-1) h(-1)) with >80% yield and >80% chromatographic purity with titers reaching 100 g L(-1). Stereochemical selectivity of DERA allows excellent enantiomeric purities (ee >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins.
authors.affiliation: Genetics, Anti-Infectives, Lek Pharmaceuticals d.d., Mengeš, Slovenia.
authors.forename: Matej
authors.initials: M
authors.lastname: Ošlaj
authors.forename: Jérôme
authors.initials: J
authors.lastname: Cluzeau
authors.forename: Damir
authors.initials: D
authors.lastname: Orkić
authors.forename: Gregor
authors.initials: G
authors.lastname: Kopitar
authors.forename: Peter
authors.initials: P
authors.lastname: Mrak
authors.forename: Zdenko
authors.initials: Z
authors.lastname: Casar
mesh_terms: D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D055162:Biocatalysis; D001711:Biotransformation; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005285:Fermentation; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones / Q000737:chemistry*
chemical_list: D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007783:Lactones; D000446:Aldehyde-Lyases; C020019:deoxyribose-phosphate aldolase