Laura Perez-Fons

Carotenoids found in Bacillus

Aims:u2002 To identify the diversity of pigmented aerobic spore formers found in the environment and to characterize the chemical nature of this pigmentation.

A genome-wide metabolomic resource for tomato fruit from Solanum pennellii

Tomato and its processed products are one of the most widely consumed fruits. Its domestication, however, has resulted in the loss of some 95% of the genetic and chemical diversity of wild relatives. In order to elucidate this diversity, exploit its potential for plant breeding, as well as understand its biological significance, analytical approaches have been developed, alongside the production of genetic crosses of wild relatives with commercial varieties. In this article, we describe a multi-platform metabolomic analysis, using NMR, mass spectrometry and HPLC, of introgression lines of Solanum pennellii with a domesticated line in order to analyse and quantify alleles (QTL) responsible for metabolic traits. We have identified QTL for health-related antioxidant carotenoids and tocopherols, as well as molecular signatures for some 2000 compounds. Correlation analyses have revealed intricate interactions in isoprenoid formation in the plastid that can be extrapolated to other crop plants.

Identification and the developmental formation of carotenoid pigments in the yellow/orange Bacillus spore-formers

Spore-forming Bacillus species capable of synthesising carotenoid pigments have recently been isolated. To date the detailed characterisation of these carotenoids and their formation has not been described. In the present article biochemical analysis on the carotenoids responsible for the yellow/orange pigmentation present in Bacilli has been carried out and the identity of the carotenoids present was elucidated. Chromatographic, UV/Vis and Mass Spectral (MS) data have revealed the exclusive presence of a C(30) carotenoid biosynthetic pathway in Bacillus species. Apophytoene was detected representing the first genuine carotenoid formed by this pathway. Cultivation in the presence of diphenylamine (DPA), a known inhibitor of pathway desaturation resulted in the accumulation of apophytoene along with other intermediates of desaturation (e.g. apophytofluene and apo-ζ-carotene). The most abundant carotenoids present in the Bacillus species were oxygenated derivatives of apolycopene, which have either undergone glycosylation and/or esterification. The presence of fatty acid moieties (C(9) to C(15)) attached to the sugar residue via an ester linkage was revealed by saponification and MS/MS analysis. In source fragmentation showed the presence of a hexose sugar associated with apolycopene derivatives. The most abundant apocarotenoids determined were glycosyl-apolycopene and glycosyl-4-methyl-apolycopenoate esters. Analysis of these carotenoids over the developmental formation of spores revealed that 5-glycosyl-4-methyl-apolycopenoate was related to sporulation. Potential biosynthetic pathways for the formation of these apocarotenoids in vegetative cells and spores have been reconstructed from intermediates and end-products were elucidated.

Combined transcript, proteome, and metabolite analysis of transgenic maize seeds engineered for enhanced carotenoid synthesis reveals pleotropic effects in core metabolism

Highlight Metabolomic, proteomic, and transcriptomic analysis of a maize line genetically engineered for enhanced seed carotenoid biosynthesis revealed how the sugar metabolism adapted to meet the additional precursor supply.

Functional characterization of long-chain prenyl diphosphate synthases from tomato

The electron transfer molecules plastoquinone and ubiquinone are formed by the condensation of aromatic head groups with long-chain prenyl diphosphates. In the present paper we report the cloning and characterization of two genes from tomato (Solanum lycopersicum) responsible for the production of solanesyl and decaprenyl diphosphates. SlSPS (S. lycopersicum solanesyl diphosphate synthase) is targeted to the plastid and both solanesol and plastoquinone are associated with thylakoid membranes. A second gene [SlDPS (S. lycopersicum solanesyl decaprenyl diphosphate synthase)], encodes a long-chain prenyl diphosphate synthase with a different subcellular localization from SlSPS and can utilize geranyl, farnesyl or geranylgeranyl diphosphates in the synthesis of C45 and C50 prenyl diphosphates. When expressed in Escherichia coli, SlSPS and SlDPS extend the prenyl chain length of the endogenous ubiquinone to nine and ten isoprene units respectively. In planta, constitutive overexpression of SlSPS elevated the plastoquinone content of immature tobacco leaves. Virus-induced gene silencing showed that SlSPS is necessary for normal chloroplast structure and function. Plants silenced for SlSPS were photobleached and accumulated phytoene, whereas silencing SlDPS did not affect leaf appearance, but impacted on primary metabolism. The two genes were not able to complement silencing of each other. These findings indicate a requirement for two long-chain prenyl diphosphate synthases in the tomato.

Biosynthesis of a novel C30 carotenoid in Bacillus firmus isolates

Pigmented Bacillus spp. with probiotic properties have been isolated. In the yellow‐/orange‐coloured strains, the carotenoid pigments present have been characterized. In contrast, the carotenoids present in the Bacillus isolates coloured red await identification. The present article reports progress on the elucidation of the pigment biosynthetic pathway in these red‐pigmented Bacillus firmus strains.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

Annotation and functional assignment of the genes for the C30 carotenoid pathways from the genomes of two bacteria: Bacillus indicus and Bacillus firmus

Bacillus indicus and Bacillus firmus synthesize C30 carotenoids via farnesyl pyrophosphate, forming apophytoene as the first committed step in the pathway. The products of the pathways were methyl 4-[6-O-acyl-glycosyl)oxy]-4,4-diapolycopen-4-oic acid and 4,4-diapolycopen-4,4-dioic acid with putative glycosyl esters. The genomes of both bacteria were sequenced, and the genes for their early terpenoid and specific carotenoid pathways annotated. All genes for a functional 1-deoxy-d-xylulose 5-phosphate synthase pathway were identified in both species, whereas genes of the mevalonate pathway were absent. The genes for specific carotenoid synthesis and conversion were found on gene clusters which were organized differently in the two species. The genes involved in the formation of the carotenoid cores were assigned by functional complementation in Escherichia coli. This bacterium was co-transformed with a plasmid mediating the formation of the putative substrate and a second plasmid with the gene of interest. Carotenoid products in the transformants were determined by HPLC. Using this approach, we identified the genes for a 4,4-diapophytoene synthase (crtM), 4,4-diapophytoene desaturase (crtNa), 4,4-diapolycopene ketolase (crtNb) and 4,4-diapolycopene aldehyde oxidase (crtNc). The three crtN genes were closely related and belonged to the crtI gene family with a similar reaction mechanism of their enzyme products. Additional genes encoding glycosyltransferases and acyltransferases for the modification of the carotenoid skeleton of the diapolycopenoic acids were identified by comparison with the corresponding genes from other bacteria.

The optimisation and application of a metabolite profiling procedure for the metabolic phenotyping of Bacillus species

The rapid advances in sequencing technologies over the last decade have enabled routine sequencing of microbial genomes. Despite notable achievements, metabolomics/metabolite profiling has not progressed with the same rapidity, which in part is due to the intrinsic complex chemical nature of the metabolome. However, well characterised metabolomes are essential if a comprehensive understanding of biological function and biotechnological applications are to be revealed and implemented. In the present study a hyphenated MS metabolite profiling procedure has been developed, predominantly for Bacillus species. The approach has been systematic in its development, delivering optimised procedures for the quenching of bacterial metabolism, extraction of metabolites, the separation and detection of components as well as data analysis, integration and visualisation workflows. Collectively, the procedure has enabled the detection of 27xa0% of the predicted Bacillus subtilis metabolome in the industrial HU36 strain. The analytical platform developed has been used to assess the chemotype of commercially used probiotic Bacillus strains, including a novel pigmented Bacillus strain HU36 that has potential either as a probiotic or source of antioxidants. The results are discussed in a biochemical context, revealing: (i), specific metabolic networks associated with pigment biosynthesis in HU36 and (ii), biotechnological applications through the demonstration of substantial equivalence.

The Formation and Sequestration of Nonendogenous Ketocarotenoids in Transgenic Nicotiana glauca

In Nicotiana glauca, plastids adapt to sequester nonendogenous carotenoids, demonstrating the robustness of plant metabolism to these changes. Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4′ β-oxygenase (crtW) and 3,3′ β-hydroxylase (crtZ) genes from Brevundimonas sp. SD-212 were expressed under constitutive transcriptional control in Nicotiana glauca, which has an emerging potential as a biofuel and biorefining feedstock. The transgenic lines produced significant levels of nonendogenous carotenoids in all tissues. In leaf and flower, the carotenoids (∼0.5% dry weight) included 0.3% and 0.48%, respectively, of nonendogenous ketolated and hydroxylated carotenoids. These were 4-ketolutein, echinenone (and its 3-hydroxy derivatives), canthaxanthin, phoenicoxanthin, 4-ketozeaxanthin, and astaxanthin. Stable, homozygous genotypes expressing both transgenes inherited the chemotype. Subcellular fractionation of vegetative tissues and microscopic analysis revealed the presence of ketocarotenoids in thylakoid membranes, not predominantly in the photosynthetic complexes but in plastoglobules. Despite ketocarotenoid production and changes in cellular ultrastructure, intermediary metabolite levels were not dramatically affected. The study illustrates the utility of Brevundimonas sp. SD-212 CRTZ and CRTW to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester nonendogenous carotenoids, and the robustness of plant metabolism to these changes.