Roles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16

Abstract

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.

FIG. 1.

R. eutropha reductase mutants exhibited different levels of PHB accumulation in both minimal and rich media. Strains were grown in fructose minimal medium, and PHB production (A) and RCDW (B) were measured. For plot A, note that the data points for the low-level-PHB-producing ΔphaB1 ΔphaB3 and ΔphaB1 ΔphaB2 ΔphaB3 strains overlap. Strains were also grown in TSB, and PHB production (C) and RCDW (D) were measured. All data points are averages from duplicate cultures.

FIG. 2.

PHB accumulation of Re2112 (ΔphaB1 ΔphaB2) changed when palm oil was used as the sole carbon source instead of fructose. Select strains were grown in palm oil minimal medium, and PHB production was measured. All data points are averages from duplicate cultures.

FIG. 3.

Addition of reductase genes to the Re2115 genome restored PHB production. H16, Re2112, and Re2115 (dashed lines) were grown at the same time as the complemented strains (solid lines) (genes added to each strain are indicated) in fructose minimal medium, and PHB production was measured. The points labeled “+phaB3” are from strain Re2143. Note that the ΔphaB1 ΔphaB2 ΔphaB3 and “+fabG” points overlap. All data points are averages from duplicate cultures.