Production of (R)-3-hydroxybutyric acid by Arxula adeninivorans

Mateusz Biernacki,Urs Hähnel,Jan Riechen,Rüdiger Bode,Gotthard Kunze,Kim Baronian,Thomas Roick

doi:10.1186/s13568-016-0303-z

Abstract

(R)-3-hydroxybutyric acid can be used in industrial and health applications. The synthesis pathway comprises two enzymes, β-ketothiolase and acetoacetyl-CoA reductase which convert cytoplasmic acetyl-CoA to (R)-3-hydroxybutyric acid [(R)-3-HB] which is released into the culture medium. In the present study we used the non-conventional yeast, Arxula adeninivorans, for the synthesis enantiopure (R)-3-HB. To establish optimal production, we investigated three different endogenous yeast thiolases (Akat1p, Akat2p, Akat4p) and three bacterial thiolases (atoBp, thlp, phaAp) in combination with an enantiospecific reductase (phaBp) from Cupriavidus necator H16 and endogenous yeast reductases (Atpk2p, Afox2p). We found that Arxula is able to release (R)-3-HB used an existing secretion system negating the need to engineer membrane transport. Overexpression of thl and phaB genes in organisms cultured in a shaking flask resulted in 4.84 g L−1 (R)-3-HB, at a rate of 0.023 g L−1 h−1 over 214 h. Fed-batch culturing with glucose as a carbon source did not improve the yield, but a similar level was reached with a shorter incubation period [3.78 g L−1 of (R)-3-HB at 89 h] and the rate of production was doubled to 0.043 g L−1 h−1 which is higher than any levels in yeast reported to date. The secreted (R)-3-HB was 99.9% pure. This is the first evidence of enantiopure (R)-3-HB synthesis using yeast as a production host and glucose as a carbon source.

Highlights

Production of chiral compounds is an area of interest for pharmaceuticals and in the fine chemicals industry
The results showed a difference between bacterial and yeast enzymes (Fig. 2a) with enzyme activities of recombinant bacterial thiolases being similar (1.38 ± 0.40, 1.37 ± 0.10 and 1.29 ± 0.05 U mg−1 for phaAp, atoBp and thlp, respectively), and while production of (R)-3-HB for G1216/YIC104-thl-phaB and G1216/YIC104-phaAphaB is comparable, the amount of (R)-3-HB secreted by G1216/YIC104-atob-phaB is significantly lower
Initial investigations, which included overexpression of different versions of thiolase genes from bacteria and A. adeninivorans and one reductase gene from C. necator H16 resulted in the production of (R)-3-HB by strains with all gene combinations, except G1216/ YIC104-AKAT4-phaB which had a mitochondrial thiolase

Summary

Introduction

Production of chiral compounds is an area of interest for pharmaceuticals and in the fine chemicals industry. A number of microbiological syntheses have been developed to produce chiral compounds (Goldberg et al 2007; Johanson et al 2005) One of these is (R)-3-hydroxybutyric acid [(R)-3-HB], which is the monomer of the polymer polyhydroxybutyrate (PHB). Matsumoto et al (2013) demonstrated production of (R)-3-HB from glucose at 5.2 g L−1 by overexpression of phaA and phaB genes from C. necator and pct, which encodes propionylCoA transferase from Clostridium propionicum, using Escherichia coli as a host organism. Employing another secretion system with tesB gene (encoding E. coli thioesterase II), Liu et al (2007) produced up to 12 g L−1 of (R)-3-HB. We describe the production of enantiopure (R)3-HB by overexpression of genes encoding β-ketothiolase and acetoacetyl-CoA reductase in the yeast A. adeninivorans

Materials and methods

Results

Findings

Discussion