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 ...

Molecular Profiling and Optimization Studies for Growth and PHB Production Conditions in Rhodobacter sphaeroides

“…The PhaZ gene, which mediates PHB depolymerization, is downregulated during the active growth phase by PhaR, a regulator protein of phaZCPR operon [5]. Recent studies revealed that disruption of the PhaZ gene and overexpression of the PhaA, PhaB, and PhaC genes elevated PHB production in R. sphaeroides [18]. Moreover, PHB biosynthetic enzymes, including PhaC, which is the limiting step for PHB production in R. sphaeroides, altered their activities very easily in response to changes in the external environment [18,50].…”

“…Among various carbon sources, acetate yielded the highest PHB production [17]. Moreover, PHB productivity can be greatly improved within R. sphaeroides, since it is possible to enhance the performance of strains using well-established genetic engineering tools and by integrating advances in previous studies [5,[18][19][20].…”

Thauera aminoaromatica MZ1T Identified as a Polyhydroxyalkanoate-Producing Bacterium within a Mixed Microbial Consortium

“…Besides PHA synthases for biopolymer production, these bacteria can produce PHA depolymerases to initiate its metabolization. To prevent PHA degradation, the PHA depolymerase(s) could be knocked out, as was done for Pseudomonas putida KT2442 and Rhodobacter sphaeroides HJ [32,33]. PHA content in cell dry mass (wt%)…”

Complete Genome Sequence of Rhodobacter sphaeroides Strain HJ, a Purple Nonsulfur Bacterium with the Ability To Produce High Levels of Hydrogen from Acetate

“…Although R. sphaeroides HJ was recently used as a host bacterium for polyhydroxyalkanoate production instead of hydrogen (2), its genome has not yet been sequenced. To make research on R.…”