title: Engineered, highly productive biosynthesis of artificial, lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed.
abstract: We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.
authors.affiliation: Lek Pharmaceuticals d.d., BU Anti Infectives, Genetics Department, Kolodvorska 27, SI-1234 Mengeš, Slovenia.
authors.forename: Tadeja
authors.initials: T
authors.lastname: Vajdič
authors.affiliation: Lek Pharmaceuticals d.d., BU Anti Infectives, Genetics Department, Kolodvorska 27, SI-1234 Mengeš, Slovenia.
authors.forename: Matej
authors.initials: M
authors.lastname: Ošlaj
authors.affiliation: Lek Pharmaceuticals d.d., BU Anti Infectives, Genetics Department, Kolodvorska 27, SI-1234 Mengeš, Slovenia.
authors.forename: Gregor
authors.initials: G
authors.lastname: Kopitar
authors.affiliation: Lek Pharmaceuticals d.d., BU Anti Infectives, Genetics Department, Kolodvorska 27, SI-1234 Mengeš, Slovenia. Electronic address: peter.mrak@sandoz.com.
authors.forename: Peter
authors.initials: P
authors.lastname: Mrak
mesh_terms: D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000378:metabolism*; D007783:Lactones / Q000378:metabolism*; D060847:Metabolic Engineering
chemical_list: D029968:Escherichia coli Proteins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007783:Lactones
keywords: Artificial biosynthetic pathway; DERA; Escherichia coli; Glucose dehydrogenase; PQQ; Pathway engineering; Respiratory chain; Super-statins; Whole-cell