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

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.

Keywords: (R)-3-HB; Acetoacetyl-CoA reductase; Arxula adeninivorans; β-Ketothiolase.

Fig. 1

Physical map of DNA transformed to auxotrophic A. adeninivorans G1216 strain for (R)-3-HB production. Fragments were obtained after linearization with AscI (fragment included d25S rDNA sequences) or SbfI (without d25S rDNA). THIOLASE is referred to atoB/thl/phaA/AKAT1/AKAT2/AKAT4/AKAT4 _N10 /AKAT4 _N17 genes encoding different versions of β-ketothiolases and REDUCTASE gene to phaB/ATPK2/AFOX2 encoding acetoacetyl-CoA reductases. Both genes were fused with TEF1 promoter and PHO5 terminator to obtain two expression modules

Fig. 2

Enzyme activities of β-ketothiolases and acetoacetyl-CoA reductase; a 24 h cultivation on YMM-glc-NO₃ (2% glucose) using all different strains, measured after 24 h; names under horizontal axis represent adequate genes overexpressed in A. adeninivorans cells; negative control—yeast transformed with empty expression plasmid. b—Enzymatic activities depends on cultivation time; G1216/YIC104-AtoB-PhaB (half-producing strain—triangle marker) and G1216/YIC104-Thl-PhaB (the best producing strain—square marker). c Enzymatic activities of different acetoacetyl-CoA reductases, usage of NADH as a cofactor instead of NADPH

Fig. 3

Time-course shaking flask culture for (R)-3-HB production; a YMM-glc-NO₃; b YPD with additional 3% of glucose; negative control—yeast transformed with empty expression plasmid; names in legends represent adequate genes overexpressed in A. adeninivorans cells and is referred to both charts

Fig. 4

(R)-3-HB utilization. After the medium shift, cultures were fed with different (R)-3-HB concentrations. Straight, dashed and dot-dashed lines correspond to the initial 0.2, 0.1 and 0% of (R)-3-HB respectively. Triangles, crosses and circles correspond to strains G1216/YIC104-thl-phaB, G1216/YIC104-phaB and G1216/YIC104 (negative control) respectively. Cell masses were shown only for the 0.2% (R)-3-HB concentration because similar growth behavior was observed for the all strains. a (R)-3-HB concentration, b cell mass (dcw)

Fig. 5

Production of (R)-3-HB by fed-batch cultivation of A. adeninivorans G1216/YIC104-Thl-PhaB: a under hypoxic condition (pO₂ = 1%) with glucose feeding; b under aerobic condition (pO₂ = 40%) with ethanol feeding. Feeding phase was started before initial glucose was completely consumed. During experiment the concentration of (R)-3-HB, glucose, ethanol and cell mass were measured