Multiscale metabolic modeling of C4 plants: connecting nonlinear genome-scale models to leaf-scale metabolism in developing maize leaves

C4 plants, such as maize, concentrate carbon dioxide in a specialized compartment surrounding the veins of their leaves to improve the efficiency of carbon dioxide assimilation. Nonlinear relationships between carbon dioxide and oxygen levels and reaction rates are key to their physiology but cannot be handled with standard techniques of constraint-based metabolic modeling. We demonstrate that incorporating these relationships as constraints on reaction rates and solving the resulting nonlinear optimization problem yields realistic predictions of the response of C4 systems to environmental and biochemical perturbations. Using a new genome-scale reconstruction of maize metabolism, we build an 18000-reaction, nonlinearly constrained model describing mesophyll and bundle sheath cells in 15 segments of the developing maize leaf, interacting via metabolite exchange, and use RNA-seq and enzyme activity measurements to predict spatial variation in metabolic state by a novel method that optimizes correlation between fluxes and expression data. Though such correlations are known to be weak in general, here the predicted fluxes achieve high correlation with the data, successfully capture the experimentally observed base-to-tip transition between carbon-importing tissue and carbon-exporting tissue, and include a nonzero growth rate, in contrast to prior results from similar methods in other systems. We suggest that developmental gradients may be particularly suited to the inference of metabolic fluxes from expression data.Comment: 57 pages, 14 figures; submitted to PLoS Computational Biology; source code available at http://github.com/ebogart/fluxtools and http://github.com/ebogart/multiscale_c4_sourc

Production of sustainable postbiotics from sugarcane straw for potential food applications

The production of postbiotics for food applications has been growing in recent years owing to their biological potential and superior technological performance over probiotics. Their production involves the use of synthetic culture media, and in this work, we propose using sugarcane straw as a source of sugar and biological components and a sustainable alternative. Thus, this by-product was used as a substrate to produce a postbiotic extract using Saccharomyces cerevisiae as a fermentative microorganism. Sugarcane straw underwent a precedent saccharification step to release the fermentable sugars. The final extracts were characterized for their total content of sugars, phenolic compounds, organic acids, and their respective chromatographic profiles. Seventeen different polyphenols were identified with the predominance of three classes, the hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids, where ferulic acid, 3,4-dihydroxybenzaldehyde, and 2,5-dihydroxybenzoic acid were most prevalent. The current work evaluated the potential use of this postbiotic extract for food applications, its antioxidant activity, gut microbiota modulatory effect, and intestinal anti-inflammatory potential. The resultant extracts showed considerable antioxidant activity and the ability to lower the pro-inflammatory mediators (i.e., interleukin 6, 8, and tumor necrosis factor-alpha) in Caco-2 cells. During the fecal fermentability assay, no modulatory effect was observed on the main beneficial bacteria, such as Lactobacillus and Bifidobacterium. Nevertheless, a significant increase in short-chain fatty acids, namely, acetate, propionate, butyrate, and valerate was observed. Moreover, the extract also demonstrated capacity to inhibit the proliferation of putrefactive bacteria such as Enterobacteriaceae. Finally, sustainable postbiotic extracts produced by S. cerevisiae fermentation using sugarcane straw as a substrate exhibited relevant biological properties with potential use as food and nutraceutical ingredients.info:eu-repo/semantics/publishedVersio

Rhodococcus opacus PD630 Genetic Tool Development to Enable the Conversion of Biomass

The discovery of fossil fuels facilitated a new era in human history and allowed many firsts, such as the mass production of goods, the ability to travel and communicate long distances, the formation of population dense cities, and unprecedented improvements in quality of life. Alternative sources of energy and chemicals are needed, however, as hydrocarbon reserves continue to deplete and the effects of burning fossils on the planet become better understood. Lignocellulosic biomass is the most abundant raw material in the world and a viable alternative to petroleum-derived products. The pre-treatment of lignocellulose (e.g., thermocatalytic depolymerization, enzymatic hydrolysis, pyrolysis, etc.) generates a range of products, including readily available sugars for microbial fermentation. One of the typically unused fractions of biomass is the structural component, referred to as lignin, that makes up 15 to 30% of the material and when depolymerized generates a heterogeneous mixture of toxic aromatic compounds. Generally, lignin is separated from the carbohydrate fraction and burned, but its utilization has been identified as a key factor in biorefinery profitability. One possible option for lignin valorization is to find a microbe that not only ferments lignocellulose-derived sugars into a valuable commodity, but also the lignin-derived aromatics.Rhodococcus opacus PD630 (hereafter R. opacus) is a non-model, gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for both lignocellulose-derived sugars and aromatic compounds, significant accumulation of the biodiesel precursor triacylglycerol, a relatively rapid growth rate, and genetic tractability. Few genetic elements and molecular biology techniques, however, have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. The goal of this dissertation is to greatly expand the genetic toolbox available in R. opacus in order to provide insight into its aromatic catabolism and to promote its use as a microbial chassis for the conversion of biomass-derived products into biofuels or other value-added products. The contributions developed as part of this dissertation include 1) the development of strong constitutive promoters for the overexpression of heterologous genes, 2) the development of chemical and metabolite sensors for tunable gene expression, 3) the characterization of native and endogenous plasmid backbones and resistance markers, 4) a heterologous T7 RNA polymerase expression platform for gene expression, 5) the demonstration of genetic logic circuits for programable gene expression, 6) a recombinase-based recombineering platform for gene knockouts and insertions, 7) a CRISPR interference (CRISPRi) platform for targeted gene repression, 8) the identification of stable reference genes for RT-qPCR applications, 9) insight into aromatic degradation through the β-ketoadipate pathway via gene knockouts, and 10) insight into the role of aromatic transporters via gene knockouts. Taken together, this work greatly advances the ability to engineer R. opacus for any desired application, in addition to providing understanding into its catabolism of aromatic compounds

Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand

Foigures 1.2, 1.4, 1.5 & 1.6 are re-used with permission.This thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism

Integration of metabolite with transcript and enzyme activity profiling during diurnal cycles in Arabidopsis rosettes

ABSTRACT: BACKGROUND: Genome-wide transcript profiling and analyses of enzyme activities from central carbon and nitrogen metabolism has shown that transcript levels undergo marked and rapid changes during diurnal cycles and after transfer to darkness, whereas changes of enzyme activities are smaller and delayed. In the starchless pgm mutant, where sugars are depleted every night, there are accentuated diurnal changes of transcript levels. Enzyme activities do not show larger diurnal changes; instead they shift towards the levels found in wild-type after several days of darkness. These results indicate that enzyme activities change slowly, integrating the changes of transcript levels over several diurnal cycles. RESULTS: To generalize this conclusion, 137 metabolites were profiled using GC-MS and LC-MS. Amplitudes of the diurnal changes of metabolites in pgm were (with the exception of sugars) similar or smaller than in wild-type. The average levels shifted towards those found after several days of darkness in wild-type. Examples include increased levels of many amino acids due to protein degradation, decreased levels of many fatty acids, increased tocopherol and decreased myo-inositol. Many metabolite-transcript correlations were found and the proportion of transcripts correlated with sugars increased dramatically in the starchless pgm mutant. CONCLUSION: Rapid diurnal changes of transcripts are integrated over time to generate quasi-stable changes across large sectors of metabolism. The slow response of enzyme activities and metabolites implies that correlations between metabolites and transcripts are due to regulation of gene expression by metabolites, rather than metabolites being changed as a consequence of a change in gene expression

Effects of must turbidity on fermentative aroma development in Sauvignon blanc

Mestrado Vinifera Euromaster - Viticulture and Enology - Instituto Superior de Agronomia - UL / Institut National d'Etudes Superieures Agronomiques de MontpellierIn many ways New Zealand has become an ideal for a New World wine country. With the first commercial Sauvignon blanc only released in 1974, Sauvignon blanc fought its way to become not only the flagship of New Zealand`s wine industry, holding around 66 % of the country’s total grape production at the 2015 harvest, but also made its way to be recognized for its outstanding quality all over the world. For some critics, New Zealand Sauvignon blanc is arguably the best in the world and others called the Marlborough wine region the best place to grow Sauvignon blanc in New Zealand. Even though testimonies like that may be subjective as taste lies on the tongue of the beholder, New Zealand Sauvignon blanc clearly came a way plastered with success leading to an export value of $1.424 billion NZD in 2015. Key to this success is that the industry found a way to distance itself from its French archetype, world famous Sauvignon blanc originated from the Loire valley, and reinvented itself, introducing a unique winestyle that does not need to shy any comparison. In the heart of this winestyle aroma compounds of the thiol group play an important role. Derived during yeast activity during fermentation from non-odorous precursors found in grapejuice, the two most important thiols that have been related with Marlborough Sauvignon blanc are 3MH and 3MHA. These deliver exotic nuances reminiscent of grapefruit, passionfruit, gooseberry, guava and boxtree. Due to the high oxidability of thiols, several strategies have been adapted by the industry to preserve these key compounds and their non-odorous precursors during the processing of grapes, which includes harvesting, transportation, processing and storage. Although a lot of research has been carried out to further understand the relations between precursors, thiols and their preserving, and to be able to continuously contrast New Zealand Sauvignon blanc from competitors, many questions still remain unclear at the present day. One of them is the effect of juice turbidity on aroma development during fermentation. Research has revealed a broad idea on the positive and negative effects, caused by micronutrients and physical parameters of grape solids derived during process steps leading to juice extraction from the berries on fermentation kinetics and general aroma development. To my best knowledge no research has been published to the present day that focuses on the effect of juice turbidity on the thiol development and expression in Sauvignon blanc ferments. This masterthesis in hand, with the title “Effects of must turbidity on fermentative aroma development in Sauvignon blanc” is supposed to give an overview over the state of research on Sauvignon blanc with a further focus on the question about possible effects and practical applications of juice turbidity. Finally, this work tries to improve the small scale winemaking protocol of Plant and Food Research, Blenheim, where this research has been conducted.N/

Plant Physiology, Development and Metabolism

Water is one of the most important constituents of life. Chemically, water is the hydride of oxygen. Oxygen, being more electronegative, exerts a strong attractive pull on its electrons. This unequal attraction results in small positive charge on twohydrogenmoleculesandasmallnegativechargeontheoxygenmolecule.The two lone pairs of electrons of the oxygen molecule result in bending of water molecule. The partial charges on oxygen and hydrogen molecules result in high electric dipole moment and polarity of water molecule

Expression of multidisciplinary flavour science : proceedings of the 12th Weurman Symposium

The 12th Weurman Flavour Research Symposium contributed 177 lectures and posters to the wealth of flavor knowledge; these were presented in eight sessions: biology, retention and release, psychophysics, quality, thermal generation, bioflavors, impact molecules, and analytics. Emerging topics were discussed in three workshops dealing with flavor and health, in vivo flavor research, and flavor metabolomics. It has been an excellent forum for passionate exchange of recent results obtained in traditional and emerging fields of flavor research. The symposium allowed coverage of the broad diversity of flavor-related topics: comprising odor and taste; applying targeted and holistic approaches; using sensorial, chemical, biological, physical, and chemometric techniques; as well as considering nutrition and health aspects

Expression of multidisciplinary flavour science : proceedings of the 12th Weurman Symposium

The 12th Weurman Flavour Research Symposium contributed 177 lectures and posters to the wealth of flavor knowledge; these were presented in eight sessions: biology, retention and release, psychophysics, quality, thermal generation, bioflavors, impact molecules, and analytics. Emerging topics were discussed in three workshops dealing with flavor and health, in vivo flavor research, and flavor metabolomics. It has been an excellent forum for passionate exchange of recent results obtained in traditional and emerging fields of flavor research. The symposium allowed coverage of the broad diversity of flavor-related topics: comprising odor and taste; applying targeted and holistic approaches; using sensorial, chemical, biological, physical, and chemometric techniques; as well as considering nutrition and health aspects