Abstract
BackgroundDue 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.ResultsWe 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.ConclusionIn 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.
Highlights
Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years
The dry cell weight (DCW) of the HJΔphaZ strain showed only a slight increase (2.02 ± 0.01 g l−1), when compared to the HJ strain (1.76 ± 0.04 g l−1), whereas the percentage of PHB content was much higher in the HJΔphaZ strain (54.2 ± 3.6%) than in its parent strain HJ (21.8 ± 3.6%) after 5 days of incubation
In this study, we found eight genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that overexpression of these genes increased PHB accumulation in a R. sphaeroides HJ strain
Summary
Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. A lot of studies on PHA production by various kinds of microorganisms have been reported so far [2,3,4,5,6] Among these microorganisms, purple non-sulfur (PNS) bacteria may be one of the most attractive candidates for PHA production improvements due to their unique characteristics. PNS bacteria can utilize light energy for potent adenosine triphosphate synthesis, and can efficiently produce various materials such as hydrogen, 5-aminolevulinic acid, and PHA at high yields. Various other growth factors were examined such as types of substrates and light intensity [10], and this study revealed that R. sphaeroides ATCC 17023 strain preferred a carbon source of about 100 mM acetate for PHB production and more than 30 μE m−2 s−1 of light intensity for anaerobic photoheterotrophic growth. Genetic differences between the wild type strain and each of the mutant strains were not analyzed
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