Heterologous Expression of Various PHA Synthase Genes in Rhodospirillum rubrum

C. Klask, M. Raberg, D. Heinrich and A. Steinbüchel

Abstract
The phototrophic non-sulfur purple bacterium Rhodospirillum rubrum is known for its metabolic versatility. Particularly, R. rubrum is able to synthesize PHA under heterotrophic or even autotrophic growth with carbon monoxide as carbon and energy source. R. rubrum has therefore become a promising candidate for future cheap PHA production. However, R. rubrum synthesizes lower amounts of PHAs in comparison to well-known PHA producers like Ralstonia eutropha H16 or recombinant Escherichia coli strains. Since the PHA synthase is the key enzyme of PHA biosynthesis, genes encoding for twelve different PHA synthases were heterologously expressed in two generated phaC deletion mutants of R. rubrum in this study. To clearly see the effect of the foreign PHA synthases, PHA-negative mutants were required. The single mutant R. rubrum ∆phaC2 showed a PHA-leaky phenotype (< 1 % PHA, wt/wt, of CDW), while the double mutant R. rubrum ∆phaC1∆phaC2 accumulated no measurable PHA. Eight different PHA synthase genes of class I, and four of class IV were chosen for heterologous expression. All recombinant R. rubrum strains showed significant PHA synthesis and accumulation, although PHA contents in the recombinant strains of the single mutant R. rubrum ∆phaC2 were generally higher in comparison to those of the double mutant R. rubrum ∆phaC1∆phaC2. Recombinant strains of the single mutant could be divided into two groups according to the accumulation of PHA in the cells. While recombinant strains dedicated to group one showed an increased PHA synthesis when compared to the wild type carrying an empty vector, strains of group two accumulated less PHA than the wild type. Finally, it was possible to increase the accumulation of PHA by up to 25 % due to heterologous expression of PHA synthase genes compared to the wild type.

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Keywords
PHB-negative deletion mutants, Rhodospirillum rubrum, Polyhydroxybutyrate (PHB), PHA-synthase genes, heterologous expression