Integration of a decrescent transcriptome and metabolomics dataset of Peucedanum praeruptorum to investigate the CYP450 and MDR genes involved in coumarins biosynthesis and transport

Abstract Peucedanum praeruptorum Dunn is well-known traditional Chinese medicine. However, little is known in the biosynthesis and the transport mechanisms of its coumarin compounds at the molecular level. Although transcriptomic sequence is playing an increasingly significant role in gene discovery, it is not sufficient in predicting the specific function of target gene. Furthermore, there is also a huge database to be analyzed. In this study, RNA sequencing assisted transcriptome dataset and high-performance liquid chromatography (HPLC) coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (Q-TOF MS)-based metabolomics dataset of P. praeruptorum were firstly constructed for gene discovery and compound identification. Subsequently, methyl jasmonate (MeJA)-induced gene expression analysis and metabolomics analysis were conducted to narrow-down the dataset for selecting the candidate genes and the potential marker metabolites. Finally, the genes involved in coumarins biosynthesis and transport were predicted with parallel analysis of transcript and metabolic profiles. As a result, a total of 40952 unigenes and 19 coumarin compounds were obtained. Based on the results of gene expression and metabolomics analysis, 7 cytochrome-P450 and 8 multidrug resistance transporter unigenes were selected as candidate genes and 8 marker compounds were selected as biomarkers, respectively. The parallel analysis of gene expression and metabolites accumulation indicated that the gene labeled as 23746, 228 and 30922 were related to the formation of the coumarin core compounds whereas 36276 and 9533 participated in the prenylation, hydroxylation, cyclization or structural modification. Similarly, 1462, 20815 and 15318 participated in the transport of coumarin core compounds while 124029 and 324293 participated in the transport of the modified compounds. This finding suggested that integration of a decrescent transcriptome and metabolomics dataset could largely narrow down the number of gene to be investigated and significantly improve the efficiency of functional gene predication. In addition, the large amount of transcriptomic data produced from P. praeruptorum and the genes discovered in this study would provide useful information in investigating the biosynthesis and transport mechanism of coumarins

Metabolic investigation in Gluconacetobacter xylinus and its bacterial cellulose production under a direct current electric field

The effects of a direct current (DC) electric field on the growth and metabolism of Gluconacetobacter xylinus were investigated in static culture. When a DC electric field at 10 mA was applied using platinum electrodes to the culture broth, bacterial cellulose (BC) production was promoted in 12 hours (h) but was inhibited in the last 12 h as compared to the control (without DC electric field). At the cathode, the presence of the hydrogen generated a strong reductive environment that is beneficial to cell growth. As compared to the control, the activities of glycolysis and tricarboxylic acid cycle, as well as BC productivity were observed to be slightly higher in the first 12 h. However, due to the absence of sufficient oxygen, lactic acid was accumulated from pyruvic acid at 18 h, which was not in favor of BC production. At the anode, DC inhibited cell growth in 6 h when compared to the control. The metabolic activity in G. xylinus was inhibited through the suppression of the tricarboxylic acid cycle and glycolysis. At 18-24 h, cell density was observed to decrease, which might be due to the electrolysis of water that significantly dropped the pH of cultural broth far beyond the optimal range. Meanwhile, metabolites for self-protection were accumulated, for instance proline, glutamic acid, gluconic acid and fatty acids. Notably, the accumulation of gluconic acid and lactic acid made it a really tough acid stress to cells at the anode and finally led to depression of cell growth

Cloning, functional characterization and catalytic mechanism of a bergaptol O-methyltransferase from Peucedanum praeruptorum Dunn

Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006) as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA). Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226 and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum

Mixed phenolic acids mediated proliferation of pathogens Talaromyces helicus and Kosakonia sacchari in continuously monocultured Radix pseudostellariae rhizosphere soil

Radix pseudostellariae L. is a common and popular Chinese medication. However, continuous monoculture has increased its susceptibility to severe diseases. We identified two pathogenic microorganisms, Talaromyces helicus M. (KU355274) and Kosakonia sacchari W. (KU324465), and their antagonistic bacterium, Bacillus pumilus Z. in rhizosphere soil of continuously monocultured R. pseudostellariae. Nine types of phenolic acids were identified both in the rhizosphere soil and in culture medium under sterile conditions. A syringic acid and phenolic acid mixture significantly promoted the growth of T. helicus and K. sacchari. T. helicus could utilize eight types of phenolic acids, whereas K. sacchari could only use four phenolic acids. K. sacchari produced protocatechuic acid when consuming vanillin. Protocatechuic acid negatively affected the growth of B. pumilus. The 3A-DON toxin produced by T. helicus promoted the growth of K. sacchari and inhibited growth of B. pumilus at low concentrations. These data help explain why phenolic exudates mediate a microflora shift and structure disorder in the rhizosphere soil of continuously monocultured R. pseudostellariae and lead to increased replanting disease incidence