Elucidation of beta-oxidation pathways in Ralstonia eutropha H16 by examination of global gene expression

Abstract

Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid β-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single β-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.

FIG. 1.

(A) Two putative fatty acid β-oxidation operons were upregulated in expression when R. eutropha H16 was grown in the presence of trioleate. Schematics 1 and 2 are two distinct gene clusters, both containing genes encoding enzymes for all reactions in the β-oxidation cycle. (B) Schematic of fatty acid β-oxidation in R. eutropha. The R. eutropha H16 gene locus tags indicate which gene products perform each step in the β-oxidation cycle. The products of four genes (A0459, transcriptional regulator; A0463, hypothetical DegV family protein; A1527, bifunctional pyrazinamidase/nicotinamidase; and A1529, phenylacetic acid degradation protein PaaI) were not assigned roles in panel B and are denoted by white arrows in panel A.

FIG. 2.

Growth of R. eutropha wild type (H16, filled triangles) and β-oxidation mutants Re2300 (ΔA0459-A0464, open squares), Re2302 (ΔA1526-A1531, filled diamonds), Re2303 (ΔA0459-A0464, A1526-A1531, open inverted triangles), and Re2312 (ΔfadD3, filled circles) in minimal medium with emulsified palm oil (A), CPKO (B), or oleic acid (C) as the carbon source. Data points are the averages of 3 separate experiments, and error bars represent the maxima and minima of each point based on 3 separate experiments.

FIG. 3.

Growth of R. eutropha wild type (H16, filled triangles) and A1322 lipase gene deletion mutant Re2313 (ΔA1322, open squares) in minimal medium with emulsified palm oil (A) or CPKO (B) as the carbon source. Data points are the average of 3 separate experiments, and error bars represent the maxima and minima of each point based on 3 separate experiments.

FIG. 4.

Growth of R. eutropha wild type (H16, filled triangles) and glyoxylate cycle mutants Re2304 (ΔaceB, open squares), Re2306 (ΔiclA, filled diamonds), and Re2307 (ΔiclB, open inverted triangles) in minimal medium with emulsified palm oil (A), CPKO (B), or oleic acid (C) as the carbon source. Data points are the averages of 3 separate experiments, and error bars represent the maxima and minima of each point based on 3 separate experiments.