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 HJΔ phaZ (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 HJΔ phaZ 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 HJΔ phaZ ( phaA3), HJΔ phaZ ( phaB2), and HJΔ phaZ ( phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔ phaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔ phaZ ( 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.

Electronic supplementary material

The online version of this article (10.1186/s12934-019-1088-y) contains supplementary material, which is available to authorized users.