Disruption of poly (3-hydroxyalkanoate) depolymerase gene and overexpression of three poly (3-hydroxybutyrate) biosynthetic genes improve poly (3-hydroxybutyrate) production from nitrogen rich medium by Rhodobacter sphaeroides

Abstract

Background: Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production.

Results: We constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJΔphaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJΔphaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJΔphaZ (phaA3), HJΔphaZ (phaB2), and HJΔphaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔphaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔphaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88 ± 0.08 g l^-1 and with 100 mM ammonium sulfate; 0.99 ± 0.05 g l^-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively.

Conclusion: In this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.

Keywords: Acetoacetyl-CoA reductase; Acetyl-CoA acetyltransferase; Poly (3-hydroxyalkanoate) depolymerase; Poly (3-hydroxyalkanoate) polymerase; Poly (3-hydroxyalkanoates); Poly (3-hydroxybutyrate); Rhodobacter sphaeroides.

Fig. 1

PHB biosynthetic pathway. First, ACAT transfers an acetyl group from one molecule of acetyl-CoA to the acetyl group of a second acetyl-CoA and produces acetoacetyl-CoA and CoASH. Second, AACR reduces the acetoacetyl group of acetoacetyl-CoA and produces (R)-3-hydroxybutanoyl-CoA. Finally, PHAP polymerizes several (R)-3-hydroxybutanoyl groups from (R)-3-hydroxybutanoyl-CoA molecules and produces PHB and CoASH. Synthesized PHB is degraded by PHADP to form (R)-3-((R)-3-hydroxybutanoyloxy) butanoate

Fig. 2

Volumetric PHB production, DCW, and PHB content of the HJ (pLP-1.2) and HJΔphaZ (pLP-1.2) strains. a Volumetric PHB production during a 5 day culturing time; b DCW after 5 days of incubation; c PHB content after 5 days of incubation. Cells were grown anaerobically at 30 °C under illumination at 8.2 W m⁻² for 5 days. Three independent fermentation experiments were done. Data are presented as the mean ± standard deviation (n = 3)

Fig. 3

Volumetric PHB production of the recombinant HJΔphaZ strains. Cells were anaerobically grown at 30 °C under illumination at 8.2 W m⁻² for 5 days. Three independent fermentation experiments were done. Data are presented as the mean ± standard deviation (n = 3)

Fig. 4

Dry cell weight and PHB content of recombinant HJΔphaZ strains. a DCW of each recombinant HJΔphaZ strain after 5 days of incubation. b PHB content of each recombinant HJΔphaZ strain after 5 days of incubation. Three independent fermentation experiments were done. Data are presented as the mean ± standard deviation (n = 3)

Fig. 5

Volumetric PHB production of the recombinant HJ and HJΔphaZ strains at different AS concentrations. a HJ (pLP-1.2) strain grown in AAY medium with various concentrations of AS. b HJΔphaZ (pLP-1.2) strain grown in AAY medium with various concentrations of AS. c HJΔphaZ (phaA3/phaB2/phaC1) strain grown in AAY medium with various concentrations of AS. Cells were grown anaerobically at 30 °C under illumination at 8.2 W m⁻² for 5 days. Three independent fermentation experiments were done. Data are presented as the mean ± standard deviation (n = 3)

Fig. 6

DCW and PHB content of the recombinant HJ and HJΔphaZ strains at different AS concentrations. a DCW of each of the recombinant HJ and HJΔphaZ strains grown in AAY medium with various concentrations of AS after 5 days of incubation. b PHB content of each of the recombinant HJ and HJΔphaZ strains grown in AAY medium with various concentrations of AS after 5 days of incubation. Cells were grown anaerobically at 30 °C under illumination at 8.2 W m⁻² for 5 days. Three independent fermentation experiments were done. Data are presented as the mean ± standard deviation (n = 3)

Fig. 7

Residual acetate concentration in the media during PHB production of recombinant HJ and HJΔphaZ strains at different AS concentrations. a HJ (pLP-1.2) strain grown in AAY medium with various concentrations of AS. b HJΔphaZ (pLP-1.2) strain grown in AAY medium with various concentrations of AS. c HJΔphaZ (phaA3/phaB2/phaC1) strain grown in AAY medium with various concentrations of AS. Cells were anaerobically grown at 30 °C under illumination at 8.2 W m⁻² for 5 days. Three independent fermentation experiments were done. Data are presented as the mean ± standard deviation (n = 3)