基于 RNA-seq 和基因缺失分析检测异养条件下 Ralstonia eutropha H16 中依赖于相位的转录组变化和 Rubisco 介导的 CO2 固定为多（3-羟基丁酸酯）。

BACKGROUND

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. 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.

RESULTS

Total 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.

CONCLUSIONS

The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.

背景

恶臭假单胞菌 H16 以高效的方式将聚羟基脂肪酸酯（PHA）作为储能物质在不平衡生长条件下产生，众所周知其可产生聚羟基脂肪酸酯（PHA），这是一种有潜力的生物基可生物降解塑料。为了进一步了解 PHA 生物合成，本研究对恶臭假单胞菌 H16 的 RNA（RNA-seq）进行了基于 cDNA 的深度测序的定量转录组分析。

结果

从果糖生长、PHA 生产和静止期的恶臭假单胞菌细胞中提取总 RNA。通过反复用针对细菌 rRNA 的磁珠处理来去除 rRNA，然后使用 Illumina 高通量测序仪确定 36 bp 序列。RNA-seq 结果表明，在生长阶段诱导了转录、翻译、细胞分裂、肽聚糖生物合成、菌毛和鞭毛组装、能量守恒和脂肪酸生物合成的基因表达；PHA 生产阶段的中心代谢相关基因表达呈下降趋势。有趣的是，即使在果糖上生长时，PHA 生产阶段 Calvin-Benson-Bassham (CBB) 循环和几个β-氧化基因的转录也明显被诱导。此外，在 NaH13CO3 的存在下，恶臭假单胞菌 H16 从果糖合成的聚（3-羟基丁酸酯）中观察到 13C 的掺入，进一步的基因缺失分析表明，CBB 循环中的两个核酮糖 1,5-二磷酸羧化酶（Rubiscos）实际上在异养条件下具有 CO2 固定功能。