Abstract
BackgroundRalstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16.ResultsTotal RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition.ConclusionsThe results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.
Highlights
Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions
This strain has been known to accumulate poly(3-hydroxybutylate) [P (3HB)] as a storage compound under unbalanced growth conditions, if a carbon source is available in excess while another essential element (N, O, P, S, or metals) is growth limiting at the same time
We investigated the possibility of CO2 fixation during P(3HB) biosynthesis by R. eutropha H16 under heterotrophic conditions, and demonstrated that both of the two ribulose 1,5-bisphosphate carboxylase (Rubisco) in the transcriptionally activated CBB cycle played a role in incorporation of 13C into P(3HB) synthesized from fructose in the presence of NaH13CO3
Summary
Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. Ralstonia eutropha H16, a Gram-negative facultative chemolithoautotrophic bacterium, can utilize various organic compounds such as sugars, organic acids, fatty acids, and plant oils in the heterotrophic growth mode, while in the absence of organic substrates, it thrives autotrophically on H2 and CO2 as the energy and carbon sources, respectively, where CO2 is fixed by CalvinBenson-Bassham (CBB) cycle [1] This strain has been known to accumulate poly(3-hydroxybutylate) [P (3HB)] as a storage compound under unbalanced growth conditions, if a carbon source is available in excess while another essential element (N, O, P, S, or metals) is growth limiting at the same time. This strain has been used as a host for metabolic engineering with the aim of biosynthesizing PHA copolyesters with more flexible properties compared with the brittle and hard P(3HB) homopolymer [9,10,11,12,13,14,15]
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