Impact of COVID-19 on national surveillance of norovirus in England and potential risk of increased disease activity in 2021.

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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

Erroneous attribution of relevant transcription factor binding sites despite successful prediction of cis-regulatory modules

<p>Abstract</p> <p>Background</p> <p><it>Cis</it>-regulatory modules are bound by transcription factors to regulate gene expression. Characterizing these DNA sequences is central to understanding gene regulatory networks and gaining insight into mechanisms of transcriptional regulation, but genome-scale regulatory module discovery remains a challenge. One popular approach is to scan the genome for clusters of transcription factor binding sites, especially those conserved in related species. When such approaches are successful, it is typically assumed that the activity of the modules is mediated by the identified binding sites and their cognate transcription factors. However, the validity of this assumption is often not assessed.</p> <p>Results</p> <p>We successfully predicted five new <it>cis</it>-regulatory modules by combining binding site identification with sequence conservation and compared these to unsuccessful predictions from a related approach not utilizing sequence conservation. Despite greatly improved predictive success, the positive set had similar degrees of sequence and binding site conservation as the negative set. We explored the reasons for this by mutagenizing putative binding sites in three <it>cis</it>-regulatory modules. A large proportion of the tested sites had little or no demonstrable role in mediating regulatory element activity. Examination of loss-of-function mutants also showed that some transcription factors supposedly binding to the modules are not required for their function.</p> <p>Conclusions</p> <p>Our results raise important questions about interpreting regulatory module predictions obtained by finding clusters of conserved binding sites. Attribution of function to these sites and their cognate transcription factors may be incorrect even when modules are successfully identified. Our study underscores the importance of empirical validation of computational results even when these results are in line with expectation.</p

Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

BACKGROUND: Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data. METHODOLOGY/PRINCIPAL FINDINGS: Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection. CONCLUSIONS/SIGNIFICANCE: Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a "bramble" model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic "root". Structural diversification may be constrained ("trimmed") where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification

Incorporation of enzyme concentrations into FBA and identification of optimal metabolic pathways

<p>Abstract</p> <p>Background</p> <p>In the present article, we propose a method for determining optimal metabolic pathways in terms of the level of concentration of the enzymes catalyzing various reactions in the entire metabolic network. The method, first of all, generates data on reaction fluxes in a pathway based on steady state condition. A set of constraints is formulated incorporating weighting coefficients corresponding to concentration of enzymes catalyzing reactions in the pathway. Finally, the rate of yield of the target metabolite, starting with a given substrate, is maximized in order to identify an optimal pathway through these weighting coefficients.</p> <p>Results</p> <p>The effectiveness of the present method is demonstrated on two synthetic systems existing in the literature, two pentose phosphate, two glycolytic pathways, core carbon metabolism and a large network of carotenoid biosynthesis pathway of various organisms belonging to different phylogeny. A comparative study with the existing extreme pathway analysis also forms a part of this investigation. Biological relevance and validation of the results are provided. Finally, the impact of the method on metabolic engineering is explained with a few examples.</p> <p>Conclusions</p> <p>The method may be viewed as determining an optimal set of enzymes that is required to get an optimal metabolic pathway. Although it is a simple one, it has been able to identify a carotenoid biosynthesis pathway and the optimal pathway of core carbon metabolic network that is closer to some earlier investigations than that obtained by the extreme pathway analysis. Moreover, the present method has identified correctly optimal pathways for pentose phosphate and glycolytic pathways. It has been mentioned using some examples how the method can suitably be used in the context of metabolic engineering.</p

Cloning and Characterization of Genes Involved in Nostoxanthin Biosynthesis of Sphingomonas elodea ATCC 31461

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2′-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2′-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed