The biosynthetic origin of psychoactive kavalactones in kava.

Kava (Piper methysticum) is an ethnomedicinal shrub native to the Polynesian islands with well-established anxiolytic and analgesic properties. Its main psychoactive principles, kavalactones, form a unique class of polyketides that interact with the human central nervous system through mechanisms distinct from those of conventional psychiatric drugs. However, an unknown biosynthetic machinery and difficulty in chemical synthesis hinder the therapeutic use of kavalactones. In addition, kava also produces flavokavains, which are chalconoids with anticancer properties structurally related to kavalactones. Here, we report de novo elucidation of the key enzymes of the kavalactone and flavokavain biosynthetic network. We present the structural basis for the evolutionary development of a pair of paralogous styrylpyrone synthases that establish the kavalactone scaffold and the catalytic mechanism of a regio- and stereo-specific kavalactone reductase that produces a subset of chiral kavalactones. We further demonstrate the feasibility of engineering styrylpyrone production in heterologous hosts, thus opening a way to develop kavalactone-based non-addictive psychiatric therapeutics through synthetic biology.

The Reference Genome Sequence of Scutellaria baicalensis Provides Insights into the Evolution of Wogonin Biosynthesis.

Scutellaria baicalensis Georgi is important in Chinese traditional medicine where preparations of dried roots, "Huang Qin," are used for liver and lung complaints and as complementary cancer treatments. We report a high-quality reference genome sequence for S. baicalensis where 93% of the 408.14-Mb genome has been assembled into nine pseudochromosomes with a super-N50 of 33.2 Mb. Comparison of this sequence with those of closely related species in the order Lamiales, Sesamum indicum and Salvia splendens, revealed that a specialized metabolic pathway for the synthesis of 4'-deoxyflavone bioactives evolved in the genus Scutellaria. We found that the gene encoding a specific cinnamate coenzyme A ligase likely obtained its new function following recent mutations, and that four genes encoding enzymes in the 4'-deoxyflavone pathway are present as tandem repeats in the genome of S. baicalensis. Further analyses revealed that gene duplications, segmental duplication, gene amplification, and point mutations coupled to gene neo- and subfunctionalizations were involved in the evolution of 4'-deoxyflavone synthesis in the genus Scutellaria. Our study not only provides significant insight into the evolution of specific flavone biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants. The reference genome of S. baicalensis is also useful for improving the genome assemblies for other members of the mint family and offers an important foundation for decoding the synthetic pathways of bioactive compounds in medicinal plants.

Genetic regulation of mitotic competence in G0 quiescent cells.

Quiescent (G0 phase) cells must maintain mitotic competence (MC) to restart the cell cycle. This is essential for reproduction in unicellular organisms and also for development and cell replacement in higher organisms. Recently, suppression of MC has gained attention as a possible therapeutic strategy for cancer. Using a Schizosaccharomyces pombe deletion-mutant library, we identified 85 genes required to maintain MC during the G0 phase induced by nitrogen deprivation. G0 cells must recycle proteins and RNA, governed by anabolism, catabolism, transport, and availability of small molecules such as antioxidants. Protein phosphatases are also essential to maintain MC. In particular, Nem1-Spo7 protects the nucleus from autophagy by regulating Ned1, a lipin. These genes, designated GZE (G-Zero Essential) genes, reveal the landscape of genetic regulation of MC.

Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast.

Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.

Complete Pathway Elucidation and Heterologous Reconstitution of Rhodiola Salidroside Biosynthesis.

Salidroside is a bioactive tyrosine-derived phenolic natural product found in medicinal plants under the Rhodiola genus. In addition to their anti-fatigue and anti-anoxia roles in traditional medicine, Rhodiola total extract and salidroside have also displayed medicinal properties as anti-cardiovascular diseases and anti-cancer agents. The resulting surge in global demand of Rhodiola plants and salidroside has driven some species close to extinction. Here, we report the full elucidation of the Rhodiola salidroside biosynthetic pathway utilizing the first comprehensive transcriptomics and metabolomics datasets for Rhodiola rosea. Unlike the previously proposed pathway involving separate decarboxylation and deamination enzymatic steps from tyrosine to the key intermediate 4-hydroxyphenylacetaldehyde (4-HPAA), Rhodiola contains a pyridoxal phosphate-dependent 4-HPAA synthase that directly converts tyrosine to 4-HPAA. We further identified genes encoding the subsequent 4-HPAA reductase and tyrosol:UDP-glucose 8-O-glucosyltransferase, respectively, to complete salidroside biosynthesis in Rhodiola. We show that heterologous production of salidroside can be achieved in the yeast Saccharomyces cerevisiae as well as the plant Nicotiana benthamiana through transgenic expression of Rhodiola salidroside biosynthetic genes. This study provides new tools for engineering sustainable production of salidroside in heterologous hosts.

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer.

Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione-related and other defense gene deletions.

Living organisms have evolved multiple sophisticated mechanisms to deal with reactive oxygen species. We constructed a collection of twelve single-gene deletion strains of the fission yeast Schizosaccharomyces pombe designed for the study of oxidative and heavy metal stress responses. This collection contains deletions of biosynthetic enzymes of glutathione (Δgcs1 and Δgsa1), phytochelatin (Δpcs2), ubiquinone (Δabc1) and ergothioneine (Δegt1), as well as catalase (Δctt1), thioredoxins (Δtrx1 and Δtrx2), Cu/Zn- and Mn- superoxide dismutases (SODs; Δsod1 and Δsod2), sulfiredoxin (Δsrx1) and sulfide-quinone oxidoreductase (Δhmt2). First, we employed metabolomic analysis to examine the mutants of the glutathione biosynthetic pathway. We found that ophthalmic acid was produced by the same enzymes as glutathione in S. pombe. The identical genetic background of the strains allowed us to assess the severity of the individual gene knockouts by treating the deletion strains with oxidative agents. Among other results, we found that glutathione deletion strains were not particularly sensitive to peroxide or superoxide, but highly sensitive to cadmium stress. Our results show the astonishing diversity in cellular adaptation mechanisms to various types of oxidative and metal stress and provide a useful tool for further research into stress responses.

Metabolism of skin-absorbed resveratrol into its glucuronized form in mouse skin.

Resveratrol (RESV) is a plant polyphenol, which is thought to have beneficial metabolic effects in laboratory animals as well as in humans. Following oral administration, RESV is immediately catabolized, resulting in low bioavailability. This study compared RESV metabolites and their tissue distribution after oral uptake and skin absorption. Metabolomic analysis of various mouse tissues revealed that RESV can be absorbed and metabolized through skin. We detected sulfated and glucuronidated RESV metabolites, as well as dihydroresveratrol. These metabolites are thought to have lower pharmacological activity than RESV. Similar quantities of most RESV metabolites were observed 4 h after oral or skin administration, except that glucuronidated RESV metabolites were more abundant in skin after topical RESV application than after oral administration. This result is consistent with our finding of glucuronidated RESV metabolites in cultured skin cells. RESV applied to mouse ears significantly suppressed inflammation in the TPA inflammation model. The skin absorption route could be a complementary, potent way to achieve therapeutic effects with RESV.

Metabolomic analysis of fission yeast at the onset of nitrogen starvation.

Microorganisms naturally respond to changes in nutritional conditions by adjusting their morphology and physiology. The cellular response of the fission yeast S. pombe to nitrogen starvation has been extensively studied. Here, we report time course metabolomic analysis during one hour immediately after nitrogen starvation, prior to any visible changes in cell morphology except for a tiny increase of cell length per division cycle. We semi-quantitatively measured 75 distinct metabolites, 60% of which changed their level over 2-fold. The most significant changes occurred during the first 15 min, when trehalose, 2-oxoglutarate, and succinate increased, while purine biosynthesis intermediates rapidly diminished. At 30-60 min, free amino acids decreased, although several modified amino acids-including hercynylcysteine sulfoxide, a precursor to ergothioneine-accumulated. Most high-energy metabolites such as ATP, S-adenosyl-methionine or NAD+ remained stable during the whole time course. Very rapid metabolic changes such as the shut-off of purine biosynthesis and the rise of 2-oxoglutarate and succinate can be explained by the depletion of NH4Cl. The changes in the levels of key metabolites, particularly 2-oxoglutarate, might represent an important mechanistic step to trigger subsequent cellular regulations.

Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

Specific biomarkers for stochastic division patterns and starvation-induced quiescence under limited glucose levels in fission yeast.

Glucose as a source of energy is centrally important to our understanding of life. We investigated the cell division-quiescence behavior of the fission yeast Schizosaccharomyces pombe under a wide range of glucose concentrations (0-111 mM). The mode of S. pombe cell division under a microfluidic perfusion system was surprisingly normal under highly diluted glucose concentrations (5.6 mM, 1/20 of the standard medium, within human blood sugar levels). Division became stochastic, accompanied by a curious division-timing inheritance, in 2.2-4.4 mM glucose. A critical transition from division to quiescence occurred within a narrow range of concentrations (2.2-1.7 mM). Under starvation (1.1 mM) conditions, cells were mostly quiescent and only a small population of cells divided. Under fasting (0 mM) conditions, division was immediately arrested with a short chronological lifespan (16 h). When cells were first glucose starved prior to fasting, they possessed a substantially extended lifespan (∼14 days). We employed a quantitative metabolomic approach for S. pombe cell extracts, and identified specific metabolites (e.g. biotin, trehalose, ergothioneine, S-adenosyl methionine and CDP-choline), which increased or decreased at different glucose concentrations, whereas nucleotide triphosphates, such as ATP, maintained high concentrations even under starvation. Under starvation, the level of S-adenosyl methionine increased sharply, accompanied by an increase in methylated amino acids and nucleotides. Under fasting, cells rapidly lost antioxidant and energy compounds, such as glutathione and ATP, but, in fasting cells after starvation, these and other metabolites ensuring longevity remained abundant. Glucose-starved cells became resistant to 40 mM H(2)O(2) as a result of the accumulation of antioxidant compounds.