A novel pre-clinical antibacterial pipeline database.

The clinical pipeline continues to be insufficient to contain antimicrobial resistance, and further investment and research is needed to ensure that a robust pipeline is built to treat the WHO priority pathogens list of antibiotic-resistant bacteria. To shed light further upstream on the preclinical pipeline the WHO has undertaken a review of the antibacterial preclinical pipeline and published the data of all identified projects in a publicly accessible database. The database captures 252 unique antibacterial agents in preclinical development being developed by 145 individual institutions, of which the majority are smaller biotech companies and academic institutions. There is a higher degree of innovation in the preclinical pipeline with a significant number of non-traditional approaches being pursued. For even a fraction of these projects to reach clinical development or the market, there is a need to shift the market dynamics for new antibacterials through the identification of new solutions beyond push and pull incentives.

Molecular characterization and safety assessment of biofortified provitamin A rice.

Part of the studies involved in safety assessment of genetically engineered crops includes characterizing the organization, integrity, and stability of the inserted DNA and evaluating the potential allergenicity and toxicity of newly-expressed proteins. Molecular characterization of the introduced DNA in provitamin A biofortified rice event GR2E confirmed insertion of a single copy of the transfer-DNA in the genome and its inheritance as a single locus. Nucleotide sequencing of the inserted DNA confirmed it was introduced without modifications. The phytoene synthase, and carotene desaturase proteins did not display sequence similarity with allergens or toxins. Both proteins were rapidly digested in simulated gastric fluid and their enzymatic activity was inhibited upon heat treatment. Acute oral toxicity testing of the protein in mice demonstrated lack of adverse effects. These evidences substantiated the lack of any identifiable hazards for both proteins and in combination with other existing comparative analyses provided assurance that food derived from this rice is safe. This conclusion is in line with those of the regulatory agencies of US Food and Drug Administration, Health Canada and Food Standard Australia and New Zealand.

Analysis of the clinical antibacterial and antituberculosis pipeline.

This analysis of the global clinical antibacterial pipeline was done in support of the Global Action Plan on Antimicrobial Resistance. The study analysed to what extent antibacterial and antimycobacterial drugs for systemic human use as well as oral non-systemic antibacterial drugs for Clostridium difficile infections were active against pathogens included in the WHO priority pathogen list and their innovativeness measured by their absence of cross-resistance (new class, target, mode of action). As of July 1, 2018, 30 new chemical entity (NCE) antibacterial drugs, ten biologics, ten NCEs against Mycobacterium tuberculosis, and four NCEs against C difficile were identified. Of the 30 NCEs, 11 are expected to have some activity against at least one critical priority pathogen expressing carbapenem resistance. The clinical pipeline is dominated by derivatives of established classes and most development candidates display limited innovation. New antibacterial drugs without pre-existing cross-resistance are under-represented and are urgently needed, especially for geographical regions with high resistance rates among Gram-negative bacteria and M tuberculosis.

Nonenzymatic ß-Carotene Degradation in Provitamin A-Biofortified Crop Plants.

Provitamin A biofortification, the provision of provitamin A carotenoids through agriculture, is regarded as an effective and sustainable intervention to defeat vitamin A deficiency, representing a global health problem. This food-based intervention has been questioned in conjunction with negative outcomes for smokers and asbestos-exposed populations of the CARET and ATBC trials in which very high doses of ß-carotene were supplemented. The current notion that ß-carotene cleavage products (apocarotenoids) represented the harmful agents is the basis of the here-presented research. We quantitatively analyzed numerous plant food items and concluded that neither the amounts of apocarotenoids nor ß-carotene provided by plant tissues, be they conventional or provitamin A-biofortified, pose an increased risk. We also investigated ß-carotene degradation pathways over time. This reveals a substantial nonenzymatic proportion of carotene decay and corroborates the quantitative relevance of highly oxidized ß-carotene polymers that form in all plant tissues investigated.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of ß-ionone ring-containing carotenes and non-epoxidated xanthophylls.

Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-ß-carotene at the C9'-C10' double bond, leading to ß-ionone and all-trans-ß-apo-10'-carotenal, both in vivo and in vitro. The enzyme also cleaved ß,ß-cryptoxanthin, zeaxanthin and lutein either at the C9'-C10' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-ß-carotene, which led to 9-cis-ß-apo-10'-carotenal. Our data indicate that all-trans-ß-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.

Transcriptional-metabolic networks in beta-carotene-enriched potato tubers: the long and winding road to the Golden phenotype.

Vitamin A deficiency is a public health problem in a large number of countries. Biofortification of major staple crops (wheat [Triticum aestivum], rice [Oryza sativa], maize [Zea mays], and potato [Solanum tuberosum]) with ß-carotene has the potential to alleviate this nutritional problem. Previously, we engineered transgenic "Golden" potato tubers overexpressing three bacterial genes for ß-carotene synthesis (CrtB, CrtI, and CrtY, encoding phytoene synthase, phytoene desaturase, and lycopene ß-cyclase, respectively) and accumulating the highest amount of ß-carotene in the four aforementioned crops. Here, we report the systematic quantitation of carotenoid metabolites and transcripts in 24 lines carrying six different transgene combinations under the control of the 35S and Patatin (Pat) promoters. Low levels of B-I expression are sufficient for interfering with leaf carotenogenesis, but not for ß-carotene accumulation in tubers and calli, which requires high expression levels of all three genes under the control of the Pat promoter. Tubers expressing the B-I transgenes show large perturbations in the transcription of endogenous carotenoid genes, with only minor changes in carotenoid content, while the opposite phenotype (low levels of transcriptional perturbation and high carotenoid levels) is observed in Golden (Y-B-I) tubers. We used hierarchical clustering and pairwise correlation analysis, together with a new method for network correlation analysis, developed for this purpose, to assess the perturbations in transcript and metabolite levels in transgenic leaves and tubers. Through a "guilt-by-profiling" approach, we identified several endogenous genes for carotenoid biosynthesis likely to play a key regulatory role in Golden tubers, which are candidates for manipulations aimed at the further optimization of tuber carotenoid content.

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta.

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

Carotenoid oxygenases involved in plant branching catalyse a highly specific conserved apocarotenoid cleavage reaction.

Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10'-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.

Biofortified crops to alleviate micronutrient malnutrition.

Micronutrient malnutrition affects more than half of the world population, particularly in developing countries. Concerted international and national fortification and supplementation efforts to curb the scourge of micronutrient malnutrition are showing a positive impact, alas without reaching the goals set by international organizations. Biofortification, the delivery of micronutrients via micronutrient-dense crops, offers a cost-effective and sustainable approach, complementing these efforts by reaching rural populations. Bioavailable micronutrients in the edible parts of staple crops at concentrations high enough to impact on human health can be obtained through breeding, provided that sufficient genetic variation for a given trait exists, or through transgenic approaches. Research and breeding programs are underway to enrich the major food staples in developing countries with the most important micronutrients: iron, provitamin A, zinc and folate.

Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway.

BACKGROUND: Since the creation of "Golden Rice", biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids. METHODOLOGY: We transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumulation of leaf carotenoids. Expression of all three genes, under tuber-specific promoter control, results in tubers with a deep yellow ("golden") phenotype without any adverse leaf phenotypes. In these tubers, carotenoids increase approx. 20-fold, to 114 mcg/g dry weight and beta-carotene 3600-fold, to 47 mcg/g dry weight. CONCLUSIONS: This is the highest carotenoid and beta-carotene content reported for biofortified potato as well as for any of the four major staple foods (the next best event being "Golden Rice 2", with 31 mcg/g dry weight beta-carotene). Assuming a beta-carotene to retinol conversion of 6ratio1, this is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A with 250 gms (fresh weight) of "golden" potatoes.

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers.

BACKGROUND: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold. RESULTS: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species. CONCLUSION: Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase.

BACKGROUND: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. RESULTS: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. CONCLUSION: The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

Why is golden rice golden (yellow) instead of red?

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.