Metabolic Engineering of Escherichia coli for Enhanced Production of ( R )- and ( S )-3-Hydroxybutyrate

Abstract

Synthetic metabolic pathways have been constructed for the production of enantiopure (R)-and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B-and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter ؊1 and 2.08 g liter ؊1 , respectively, in shake flask cultures within 2 days. The NADPH/NADP ؉ ratio was found to be two-to three-fold higher than the NADH/NAD ؉ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…The reduction in titre is likely due to the change from an NADPH-dependent dehydrogenase (Adh6p Sc ) to an NADHdependent dehydrogenase (Lsadh Ls ) under the aerobic conditions used. The ratio of NADH/NAD þ has been observed to be lower than that of NADPH/NAD þ under similar culture conditions 64 .…”

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

“…It is well documented that PhaB results exclusively in (R)-3-hydroxyacids while Hbd results in the (S) stereoisomers 33,48 . This phenomenon was experimentally verified in pathways producing both 3HB 29 and 3HV 30 . Because 3,4-DHBA has a hydroxyl group in the d-position that changes the stereochemical priority of the different atoms of the molecule about its stereocenter, the stereochemistry of 3,4-DHBA formed by PhaB should be (S)-3,4-DHBA while that formed by Hbd should be (R)-3,4-DHBA.…”

“…PCR products were digested with the appropriate restriction enzymes and ligated directly into similarly digested vectors. The ptb-buk fragment was generated by EcoRI and NotI digestion of pCDF-PB 29 . The B. subtilis ptb and buk genes were cloned into an artificial operon using splicing by overlap extension PCR 54,55 to mimic the natural C. acetobutylicum ptb-buk operon.…”

“…Although many substituent monomers have been incorporated into PHAs through modulation of substrates provided in the culture medium 26,27 , the number of monomers produced directly from simple carbon sources is far smaller. Indeed, only 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) have been successfully produced at the gram per litre scale using enzymes of the PHA biosynthetic pathway [28][29][30] . In these reports, 3HB or 3HV synthesis begins with the condensation of two acetyl-CoA molecules or acetyl-CoA and propionyl-CoA, respectively, through the action of a thiolase enzyme (PhaA or BktB).…”

“…The b-keto group is subsequently reduced to an alcohol by one of the two 3-hydroxybutyryl-CoA dehydrogenases, PhaB or Hbd, yielding the (R) or (S)-enantiomer, respectively. Lastly, production of the free acid requires hydrolysis of the CoA moiety, which can be accomplished by the enzyme pair phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) 28,31 , or by thioesterase II (TesB) 29,32,33 . TesB is capable of hydrolyzing both enantiomers of 3HB-CoA, whereas the Ptb-Buk enzyme system is specific to the (R)-enantiomer.…”