Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae

Abstract

Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJ(Ac). Escherichia coli BL21(DE3) carrying phaJ(Ac). under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ(Ac) except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJ(Ac) exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ(Ac) is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid beta-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae.

FIG. 1

(a) Schematic drawing of a 3.2-kbp EcoRI fragment containing ORF1, phaC_Ac, and phaJ_Ac (ORF3) with a putative promoter region from A. caviae FA440. (b) The ability of PJRDEE32 and its deleted clones to complement a PHA-negative mutant of A. caviae (AC004). PHA production was carried out on 1% dodecanoate by two-step fermentation, as described in the text.

FIG. 2

Construction of plasmid pETNB3 for overexpression of phaJ_Ac in E. coli BL21(DE3). A designed ribosome binding site (RBS) from pET-3a is indicated (underlined). P_T7, T7 promoter in pET-3a.

FIG. 3

SDS-PAGE analysis of enoyl-CoA hydratase from E. coli BL21(DE3)/pETNB3. Lanes: 1, molecular mass standard proteins, with the masses indicated on the left (from top to bottom, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor); 2, crude extract proteins of E. coli BL21(DE3)/pETNB3; 3, purified enoyl-CoA hydratase after chromatography on Q-Sepharose.

FIG. 4

Elution profile of enoyl-CoA hydratase from E. coli BL21(DE3)/pETNB3. The soluble protein fraction from the cells grown in four 100-ml cultures was applied to a Q-Sepharose column. ░⃞, absorbance at 280 nm; ——, concentration of NaCl; •, enoyl-CoA hydratase activity toward crotonyl-CoA.

FIG. 5

Evaluation of stereospecificity of enoyl-CoA hydratase encoded by phaJ_Ac. (a) Hydration of crotonyl-CoA coupled with (S)-specific dehydrogenation of 3HB-CoA catalyzed by (S)-3HA-CoA dehydrogenase. The reaction mixture was composed of 0.25 mM crotonyl-CoA, 0.5 mM NAD⁺, 6 mU of (S)-3HA-CoA dehydrogenase, and 1 U of hydratase in 400 μl of 50 mM Tris-HCl (pH 8.0). (b) Hydration of crotonyl-CoA coupled with (R)-specific polymerization of 3HB-CoA catalyzed by crude PHA synthase. The reaction mixture was composed of 0.25 mM crotonyl-CoA, 10 mM DTNB, 1.2 mU of PHA synthase (a crude extract of A. eutrophus H16 containing 10 μg of protein), and 1 U of hydratase in 400 μl of 125 mM potassium phosphate (pH 7.2). The crude extract of A. eutrophus H16 was prepared from cells grown on fructose for 30 h at 30°C, as described previously (25). Symbols: ○, addition of phaJ_Ac-derived enoyl-CoA hydratase; □, addition of crotonase [(S)-specific enoyl-CoA hydratase]; ▴, no addition of enoyl-CoA hydratase.

FIG. 6

Proposed pathway of P(3HB-co-3HHx) biosynthesis by A. caviae from alkanoic acids or oils. 1, β-ketothiolase; 2, NADH-acetoacetyl-CoA dehydrogenase; 3, crotonase [(S)-specific enoyl-CoA hydratase].