A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

Abstract

Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65-70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn²⁺ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h^-1, and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

Figure 1

Scheme of the anabolic and catabolic pathways starting with pyruvate. In the figure: AHAS, anabolic acetohydroxyacid synthase; cALS, catabolic α-acetolactate synthase; ALDC, α-acetolactate decarboxylase; BDH, 2,3-butanediol dehydrogenase; and AR, diacetyl reductase.

Figure 2

SDS-PAGE analysis of the recombinant enzymes involved in converting pyruvate to acetoin.

Figure 3

Temperature and pH profiles of the recombinant enzymes involved in converting pyruvate to acetoin. (A) Optimum temperatures of coAHASL1, coAHASL2 and bsALDC. (B) Optimum pH values of coAHASL1, coAHASL2 and bsALDC. Error bars indicate standard deviations of three independent experiments.

Figure 4

Optimization of the cell-free biosynthetic system. (A) Effect of the enzyme loading ratio on acetoin synthesis. Determination of the effect of the enzyme ratio on the synthesis of acetoin was tested in a total enzyme loading of 0.1 U mL⁻¹ with 10 mM pyruvate. The ratio between coAHASL1 and bsALDC was changed from 0.25 to 4. (B) Effect of substrate loading on acetoin synthesis. Determination of the effect of the substrate concentration on the synthesis of acetoin was carried out by changing the concentration of sodium pyruvate from 5 to 50 mM. The total enzyme (coAHASL1/bsALDC 4:1) was applied at a ratio of 1 U/10 mM substrate. All reactions were conducted at 65 °C in citrate-phosphate buffer (pH = 6.5) for 12 h. Error bars indicate standard deviations of three independent experiments.

Figure 5

Production of acetoin from pyruvate under the optimal conditions. (A) Time course of the biocatalysis. (B) HPLC analysis of the products after reaction for 24 h. The reaction mixture contained 10 mM sodium pyruvate, 1 U total enzyme (coAHASL1/bsALDC 4:1), 0.5 mM ThDP, 10 μM FAD, 10 mM MgC1₂ and 10 mM MnC1₂. The reaction was conducted at 65 °C in 10 mL of citrate-phosphate buffer (pH = 6.5). Error bars indicate standard deviations of three independent experiments.