Plants Metabolome Study: Emerging Tools and Techniques

Metabolomics is now considered a wide-ranging, sensitive and practical approach to acquire useful information on the composition of a metabolite pool present in any organism, including plants. Investigating metabolomic regulation in plants is essential to understand their adaptation, acclimation and defense responses to environmental stresses through the production of numerous metabolites. Moreover, metabolomics can be easily applied for the phenotyping of plants; and thus, it has great potential to be used in genome editing programs to develop superior next-generation crops. This review describes the recent analytical tools and techniques available to study plants metabolome, along with their significance of sample preparation using targeted and non-targeted methods. Advanced analytical tools, like gas chromatography-mass spectrometry (GC-MS), liquid chromatography mass-spectroscopy (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) matrix-assisted laser desorption/ionization (MALDI), ion mobility spectrometry (IMS) and nuclear magnetic resonance (NMR) have speed up precise metabolic profiling in plants. Further, we provide a complete overview of bioinformatics tools and plant metabolome database that can be utilized to advance our knowledge to plant biology.

Pyridoxal and α-ketoglutarate independently improve function of Saccharomyces cerevisiae Thi5 in the metabolic network of Salmonella enterica

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica . Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report a strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica . Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica . IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contribute to their functional differences in a Salmonella enterica host.

Plants Metabolome Study: Emerging Tools and Techniques

Metabolomics is now considered to be a wide-ranging, sensitive and practical approach to acquire useful information on the composition of a metabolite pool present in any organism, including plants. Investigating metabolomic regulation in plants is essential to understand their adaptation, acclimation and defense response to environmental stresses through the production of numerous metabolites. Moreover, metabolomics can be easily applied for the phenotyping of plants; and thus, it has great potential to be used in molecular breeding and genome editing programs to develop superior next generation crops. This review describes the recent analytical tools and techniques available to study plants metabolome, along with their significance of sample preparation using targeted and non-targeted method. Advanced analytical tools, like gas chromatography-mass spectrometry (GC-MS), liquid chromatography mass-spectroscopy (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) and matrix-assisted laser desorption/ionization (MALDI) have speed up metabolic profiling in plants. Further, we deliver a complete overview of bioinformatics tools and plant metabolome database that can be utilized to advance our knowledge to plant biology.

Microbial cell factories for the production of polyhydroxyalkanoates

Pollution caused by persistent petro-plastics is the most pressing problem currently, with 8 million tons of plastic waste dumped annually in the oceans. Plastic waste management is not systematized in many countries, because it is laborious and expensive with secondary pollution hazards. Bioplastics, synthesized by microorganisms, are viable alternatives to petrochemical-based thermoplastics due to their biodegradable nature. Polyhydroxyalkanoates (PHAs) are a structurally and functionally diverse group of storage polymers synthesized by many microorganisms, including bacteria and Archaea. Some of the most important PHA accumulating bacteria include Cupriavidus necator, Burkholderia sacchari, Pseudomonas sp., Bacillus sp., recombinant Escherichia coli, and certain halophilic extremophiles. PHAs are synthesized by specialized PHA polymerases with assorted monomers derived from the cellular metabolite pool. In the natural cycle of cellular growth, PHAs are depolymerized by the native host for carbon and energy. The presence of these microbial PHA depolymerases in natural niches is responsible for the degradation of bioplastics. Polyhydroxybutyrate (PHB) is the most common PHA with desirable thermoplastic-like properties. PHAs have widespread applications in various industries including biomedicine, fine chemicals production, drug delivery, packaging, and agriculture. This review provides the updated knowledge on the metabolic pathways for PHAs synthesis in bacteria, and the major microbial hosts for PHAs production. Yeasts are presented as a potential candidate for industrial PHAs production, with their high amenability to genetic engineering and the availability of industrial-scale technology. The major bottlenecks in the commercialization of PHAs as an alternative for plastics and future perspectives are also critically discussed.

Seagrass Metabolomics: A New Insight towards Marine Based Drug Discovery

Metabolomics is one of the new field of “Omics” approach and the youngest triad of system biology, which provides a broad prospective of how metabolic networks are controlled and indeed emerged as a complementary tool to functional genomics with well-established technologies for genomics, transcriptomics and proteomics. Though, metabolite profiling has been carried out for decades, owing to decisive mechanism of a molecule regulation, the importance of some metabolites in human regimen and their use as diagnostic markers is now being recognized. Plant metabolomics therefore aims to highlight the characterization of metabolite pool of a plant tissue in response to its environment. Seagrassses, a paraphyletic group of marine hydrophilous angiosperms which evolved three to four times from land plants back to the sea. Seagrasses share a number of analogous acquired metabolic adaptations owing to their convergent evolution, but their secondary metabolism varied among the four families that can be considered as true seagrasses. From a chemotaxonomic point of view, numerous specialized metabolites have often been studied in seagrasses. Hence, this chapter focus the metabolome of seagrasses in order to explore their bioactive properties and the recent advancements adopted in analytical technology platforms to study the non-targeted metabolomics of seagrasses using OMICS approach.

Plant Metabolomics: An Overview

The term metabolomics was coined by Oliver and his group in 1998. It is a study of biochemical profile and regulation of functions in whole organism by analyzing a metabolite pool present in organism. Researchers believe that more than 400,000 plant species exist worldwide. Total number of metabolites in the plant kingdom are about 2,00,000 to 10,00,000. With the availability of highly sensitive and selective analytical techniques, metabolic changes in plant systems can be followed in a comprehensive way. This technology is useful in assessing gene function and relationships to metabolites. The nutritional values of food and concentration of pharmaceuticals in plants can be improved by using metabolomics study and its functional genomic strategies. Metabolomics analysis is comparatively fast, cheaper and reliable, but simultaneous identification of all metabolites in a crop plant remains a challenge.

Phycobilisome breakdown effector NblD is required to maintain the cellular amino acid composition during nitrogen starvation

Nitrogen starvation induces a coordinated nitrogen-saving program in cyanobacteria. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the ornithine-ammonia cycle and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD-deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, carbon fixation because the pool of 3-phosphoglyceric acid, the stable CO2 fixation product of RubisCO, was also decreased, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite coregulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply.

Impact of Altered Trehalose Metabolism on Physiological Response of Penicillium chrysogenum Chemostat Cultures during Industrially Relevant Rapid Feast/Famine Conditions

Due to insufficient mass transfer and mixing issues, cells in the industrial-scale bioreactor are often forced to experience glucose feast/famine cycles, mostly resulting in reduced commercial metrics (titer, yield and productivity). Trehalose cycling has been confirmed as a double-edged sword in the Penicillium chrysogenum strain, which facilitates the maintenance of a metabolically balanced state, but it consumes extra amounts of the ATP responsible for the repeated breakdown and formation of trehalose molecules in response to extracellular glucose perturbations. This loss of ATP would be in competition with the high ATP-demanding penicillin biosynthesis. In this work, the role of trehalose metabolism was further explored under industrially relevant conditions by cultivating a high-yielding Penicillium chrysogenum strain, and the derived trehalose-null strains in the glucose-limited chemostat system where the glucose feast/famine condition was imposed. This dynamic feast/famine regime with a block-wise feed/no feed regime (36 s on, 324 s off) allows one to generate repetitive cycles of moderate changes in glucose availability. The results obtained using quantitative metabolomics and stoichiometric analysis revealed that the intact trehalose metabolism is vitally important for maintaining penicillin production capacity in the Penicillium chrysogenum strain under both steady state and dynamic conditions. Additionally, cells lacking such a key metabolic regulator would become more sensitive to industrially relevant conditions, and are more able to sustain metabolic rearrangements, which manifests in the shrinkage of the central metabolite pool size and the formation of ATP-consuming futile cycles.

Adaptation of Central Metabolite Pools to Variations in Growth Rate and Cultivation Conditions in Saccharomyces Cerevisiae

Background: Saccharomyces cerevisiae is a well-known popular model system for basic biological studies and to serve as host organism for heterologous production of commercially interesting small molecules and proteins. The central metabolism is at the core to provide building blocks and energy to support growth and survival in normal situations as well as during exogeneous stresses and forced heterologous protein production. Here, we present a comprehensive study of intracellular central metabolite pool profiling when growing S. cerevisiae on different carbon sources in batch cultivations and at different growth rates in nutrient limited glucose chemostats. Latest versions of absolute quantitative mass spectrometry-based metabolite profiling methodology were applied to cover glycolytic and pentose phosphate pathway metabolites, TCA, complete amino acid and deoxy-/nucleoside phosphate pools. We have attempted to correlate the total metabolite pool composition with growth rates and nutrient limitation in both batch and chemostat cultivations. We have also tried to dissect the Crabtree-effect, i.e. ethanol-producing cultivation conditions, based on metabolite pool composition. Results: Glutamate, glutamine, alanine and citrate were the four most abundant metabolites for most conditions tested. Amino acid is the dominant metabolite class even though a marked relative reduction compared to the other metabolite classes was observed for nitrogen and phosphate limited chemostats. Interestingly, glycolytic and PPP metabolites display largest variation among the cultivation conditions while the nucleoside phosphate pools are more stable and vary within a closer concentration window. The overall trends for glucose and nitrogen limited chemostats were increased metabolite pools with increasing growth rate. Next, comparing the chosen chemostat reference growth rate (0.12 h -1 , approximate one-fourth of maximal unlimited growth rate) illuminates an interesting pattern: almost all pools are lower in nitrogen and phosphate limited conditions compared to glucose limitation, except for the TCA metabolites citrate, isocitrate and a-ketoglutarate. Conclusions: This study provides new knowledge how the central metabolism is adapting to various cultivations conditions and growth rates which is essential for expanding our understanding of cellular metabolism and development of improved phenotypes in metabolic engineering.