Horizontal Gene Transfer and Fusion Spread Carotenogenesis Among Diverse Heterotrophic Protists.

Thraustochytrids (phylum: Labyrinthulomycota) are nonphotosynthetic marine protists. Some thraustochytrids have crtIBY, a trifunctional fusion gene encoding a protein capable of ß-carotene biosynthesis from geranylgeranyl pyrophosphate. Here we show that crtIBY is essential in, and encodes the sole pathway for, carotenoid biosynthesis in the thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381. We explore the evolutionary origins of CrtIBY and discover that the closest related protein domains are present in a small but diverse group of other heterotrophic protists, including the apusomonad Thecamonas trahens and the dinoflagellates Oxyrrhis marina and Noctiluca scintillans. Each organism within this cluster also contains one or more ß-carotene 15-15' oxygenase genes (blh and rpe65), suggesting that the acquisition of ß-carotene biosynthesis genes may have been related to the production of retinal. Our findings support a novel origin of eukaryotic (apo)carotenoid biosynthesis by horizontal gene transfer from Actinobacteria, Bacteroidetes, and/or Archaea. This reveals a remarkable case of parallel evolution of eukaryotic (apo)carotenogenesis in divergent protistan lineages by repeated gene transfers.

Golden Gate Multigene Assembly Method for Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica has emerged as a powerful alternative for biolipid production due to its high capacity for lipid accumulation. Genetic engineering and synthetic biology are promoted forward to improve production and reroute metabolism for high-value compound synthesis. In this context, efficient, modular, and high-throughput compatible cloning and expression system are required to speed up and rationalize research in this field. Here, we present the fast and modular Golden Gate cloning strategy for the construction of multigene expression vectors and their transformation into Y. lipolytica. As an example, we used the heterologous expression of the carotenoid pathway by cloning three genes involved in this pathway in only one vector allowing reaching production of ß-carotene after a single transformation.

GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous.

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one-hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two-hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild-type strain did not largely modify the carotenoid profile, indicating translational and/or post-translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild-type strain and the XdWC2-disrupted strain apparently improved the production of monocyclic carotenoid 3-hydroxy-3', 4'-didehydro-ß, ψ-carotene-4-one (HDCO) rather than ß-carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over-producing strains.

Development of ß-carotene, lysine, and tryptophan-rich maize (Zea mays) inbreds through marker-assisted gene pyramiding.

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased ß-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in ß-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1-BC3F1, and ICF1-ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3'TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45-88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the ß-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-ß1σ4-4-8-8, DBT-IC-ß1σ4-9-21-21, DBT-IC-ß1σ4-10-1-1, DBT-IC-ß2σ5-9-51-51, DBT-IC-ß2σ5-9-52-52 and DBT-IC-ß2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78-99.31% for DBT-IC-ß1σ4 population and 85.71-99.51% for DBT-IC-ß2σ5 population) like the recurrent parents and ß-carotene (14.21-14.35 µg/g for DBT-IC-ß1σ4 and 13.28-13.62 µg/g for DBT-IC-ß2σ5), lysine (0.31-0.33% for DBT-IC-ß1σ4 and 0.31-0.34% for DBT-IC-ß2σ5), and tryptophan (0.079-0.082% for DBT-IC-ß1σ4 and 0.078-0.083% for DBT-IC-ß2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs.

Genome-Wide Identification of the B-Box Gene Family and Expression Analysis Suggests Their Potential Role in Photoperiod-Mediated ß-Carotene Accumulation in the Endocarp of Cucumber (Cucumis sativus L.) Fruit.

Carotenoids are indispensable to plants and essential for human nutrition and health. Carotenoid contents are strongly influenced by light through light-responsive genes such as B-Box (BBX) genes. BBX proteins, a class of zinc-finger transcription factors, mediate many light-signaling pathways, leading to the biosynthesis of important metabolites in plants. However, the identification of the BBX gene family and expression analysis in response to photoperiod-mediated carotenoid accumulation in cucumber remains unexplored. We performed a genome-wide study and determined the expression of cucumber BBX genes (hereafter referred to as CsaBBXs genes) in the endocarp of Xishuangbanna cucumber fruit (a special type of cucumber accumulating a high level of ß-carotene in the endocarp) using an RNA-seq analysis of plants previously subjected to two photoperiodic conditions. Here, 26 BBX family genes were identified in the cucumber genome and named serially CsaBBX1 through CsaBBX26. We characterized CsaBBX genes in terms of their phylogenetic relationships, exon-intron structures, cis-acting elements, and syntenic relationships with Arabidopsis thaliana (L.) Heynh. RNA-seq analysis revealed a varied expression of CsaBBX genes under photoperiod treatment. The analysis of CsaBBXs genes revealed a strong positive correlation between CsaBBX17 and carotenoid biosynthetic pathway genes (phytoene synthase, ζ-carotene desaturase, lycopene ε-cyclase, ß-carotene hydroxylase-1), thus suggesting its involvement in ß-carotene biosynthesis. Additionally, nine CsaBBX genes (CsaBBX 4,5,7,9,11, 13,15,17 and 22) showed a significant positive correlation with ß-carotene content. The selected CsaBBX genes were verified by qRT-PCR and confirmed the validity of RNA-seq data. The results of this study established the genome-wide analysis of the cucumber BBX family and provide a framework for understanding their biological role in carotenoid accumulation and photoperiodic responses. Further investigations of CsaBBX genes are vital since they are promising candidate genes for the functional analysis of carotenoid biosynthesis and can provide genetic tools for the molecular breeding of carotenoids in plants.

Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved ß-carotene production in Saccharomyces cerevisiae.

BACKGROUND: The limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve ß-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches. RESULTS: The direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of ß-carotene in S. cerevisiae. However, due to the competition for precursor between ß-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on ß-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in ß-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to ß-carotene pathway and achieved additional 31.7% increase in ß-carotene content without adversely affecting cell growth. By inducing an extra constitutive ß-carotene synthesis pathway for further conversion precursor farnesol to ß-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of ß-carotene, which is 107.3% and 49.5% increase respectively over the parent strain. CONCLUSIONS: This strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.

Mutant combinations of lycopene ɛ-cyclase and ß-carotene hydroxylase 2 homoeologs increased ß-carotene accumulation in endosperm of tetraploid wheat (Triticum turgidum L.) grains.

Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein-with low natural variation in provitamin A ß-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ɛ-cyclase (LCYe) and ß-carotene hydroxylase (HYD) function in diverting carbons from ß-carotene to lutein biosynthesis and catalyzing the turnover of ß-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase ß-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased ß-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent ß-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that ß-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.

AgZDS, a gene encoding ζ-carotene desaturase, increases lutein and ß-carotene contents in transgenic Arabidopsis and celery.

ζ-Carotene desaturase (ZDS) is one of the key enzymes regulating carotenoids biosynthesis and accumulation. Celery transgenic efficiency is low and it is difficult to obtain transgenic plants. The study on ZDS was limited in celery. Here, the AgZDS gene was cloned from celery and overexpressed in Arabidopsis thaliana and celery to verify its function. The AgZDS has typical characteristic of ZDS protein and is highly conserved in higher plants. Phylogenetic analysis showed that AgZDS has the closest evolutionary relationship with ZDSs from Solanum lycopersicum, Capsicum annuum and Tagetes erecta. Overexpression of AgZDS gene in A. thaliana and celery resulted in increased accumulations of lutein and ß-carotene and up-regulated the expression levels of the genes involved in carotenoids biosynthesis. The contents of lutein and ß-carotene in two lines, AtL1 and AgL5, were the highest in transgenic A. thaliana and celery, respectively. The relative expression levels of 5 genes (AtPDS, AtZISO, AtZEP, AtNCED3, and AtCCD4) were up-regulated compared to the wild type plants. The relative expression levels of most genes in carotenoids biosynthesis pathway, such as AgPDS, AgCRTISO1, and AgZISO, were up-regulated in transgenic celery plants. The antioxidant capacity of A. thaliana and photosynthetic capacity of celery were also enhanced. This research is the first report on the function of structure gene related to carotenoid biosynthesis in transgenic celery plants. The findings in this study demonstrated the roles of AgZDS in regulating carotenoids metabolism of celery, which laid a potential foundation for quality improvement of celery.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for ß-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in ß-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285ß+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the ß-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74-90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and ß-carotene content. The agronomic performance of the four lines was similar (78.83-99.44%) to that of the recurrent parent, and ß-carotene content (7.541-8.711 µg/g) was on par with the donor parent. Our study is the first to improve the ß-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.

Robust and flexible platform for directed evolution of yeast genetic switches.

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene.

Chlamydomonas reinhardtii is a well-established microalgal model species with a shorter doubling time, which is a promising natural source for the efficient production of high-value carotenoids. In the microalgal carotenoid biosynthetic pathway, lycopene is converted either into ß-carotene by lycopene ß-cyclase or into α-carotene by lycopene ε-cyclase (LCYE) and lycopene ß-cyclase. In this study, we overexpressed the LCYE gene in C. reinhardtii to estimate its effect on lycopene metabolism and lutein production. Chlamydomonas transformants (CrLCYE#L1, #L5, and #L6) produced significantly increased amounts of lutein per culture (up to 2.6-fold) without a decrease in cell yields. Likewise, the expression levels of LCYE gene in transformants showed a significant increase compared with that of the wild-type strain. These results suggest that LCYE overexpression enhances the conversion of lycopene to α-carotene, which in turn improves lutein productivity. Interestingly, their ß-carotene productivity appeared to increase slightly rather than decrease. Considering that the inhibition of the lycopene cyclization steps often induces higher expression in genes upstream of metabolic branches, this result implies that the redirection from ß-carotene to α-carotene by LCYE overexpression might also enhance upstream gene expression, thereby leading to auxiliary ß-carotene production.

Development and characterization of GR2E Golden rice introgression lines.

Golden Rice with ß-carotene in the grain helps to address the problem of vitamin A deficiency. Prior to commercialize Golden Rice, several performance and regulatory checkpoints must be achieved. We report results of marker assisted backcross breeding of the GR2E trait into three popular rice varieties followed by a series of confined field tests of event GR2E introgression lines to assess their agronomic performance and carotenoid expression. Results from confined tests in the Philippines and Bangladesh have shown that GR2E introgression lines matched the performance of the recurrent parents for agronomic and yield performance, and the key components of grain quality. Moreover, no differences were observed in terms of pest and disease reaction. The best performing lines identified in each genetic background had significant amounts of carotenoids in the milled grains. These lines can supply 30-50% of the estimated average requirements of vitamin A.

Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates.

ß-Carotene is a yellow-orange-red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for ß-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of ß-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding ß-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L ß-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest ß-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens-mediated transformation method.

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

Light dependent accumulation of ß-carotene enhances photo-acclimation of Euglena gracilis.

Carotenoids are essential components of photosynthetic organisms including land plants, algae, cyanobacteria, and photosynthetic bacteria. Although the light-mediated regulation of carotenoid biosynthesis, including the light/dark cycle as well as the dependence of carotenoid biosynthesis-related gene translation on light wavelength, has been investigated in land plants, these aspects have not been studied in microalgae. Here, we investigated carotenoid biosynthesis in Euglena gracilis and found that zeaxanthin accumulates in the dark. The major carotenoid species in E. gracilis, namely ß-carotene, neoxanthin, diadinoxanthin and diatoxanthin, accumulated corresponding to the duration of light irradiation under the light/dark cycle, although the translation of carotenoid biosynthesis genes hardly changed. Irradiation with either blue or red-light (3 µmol photons m-2 s-1) caused a 1.3-fold increase in ß-carotene content compared with the dark control. Blue-light irradiation (300 µmol photons m-2 s-1) caused an increase in the cellular content of both zeaxanthin and all trans-diatoxanthin, and this increase was proportional to blue-light intensity. In addition, pre-irradiation with blue-light of 3 or 30 µmol photons m-2 s-1 enhanced the photosynthetic activity and tolerance to high-light stress. These findings suggest that the accumulation of ß-carotene is regulated by the intensity of light, which may contribute to the acclimation of E. gracilis to the light environment in day night conditions.

Genetically Modified Plants: Nutritious, Sustainable, yet Underrated.

Combating malnutrition is one of the greatest global health challenges. Plant-based foods offer an assortment of nutrients that are essential for adequate nutrition and can promote good health. Unfortunately, the majority of widely consumed crops are deficient in some of these nutrients. Biofortification is the umbrella term for the process by which the nutritional quality of food crops is enhanced. Traditional agricultural breeding approaches for biofortification are time consuming but can enhance the nutritional value of some foods; however, advances in molecular biology are rapidly being exploited to biofortify various crops. Globally, genetically modified organisms are a controversial topic for consumers and governmental agencies, with a vast majority of people apprehensive about the technology. Golden Rice has been genetically modified to contain elevated ß-carotene concentrations and is the bellwether for both the promise and angst of agricultural biotechnology. Although there are numerous other nutritional targets of genetically biofortified crops, here I briefly summarize the work to elevate iron and folate concentrations. In addition, the possibility of using modified foods to affect the gut microbiota is examined. For several decades, plant biotechnology has measured changes in nutrient concentrations; however, the bioavailability of nutrients from many biofortified crops has not been demonstrated.

A multifocal approach towards understanding the complexities of carotenoid biosynthesis and accumulation in rice grains.

Carotenoids are mostly C40 terpenoids that participate in several important functions in plants including photosynthesis, responses to various forms of stress, signal transduction and photoprotection. While the antioxidant potential of carotenoids is of particular importance for human health, equally important is the role of ß-carotene as the precursor for vitamin A in the human diet. Rice, which contributes upto 40% of dietary energy for mankind, contains very low level of ß-carotene, thereby making it an important crop for enhancing ß-carotene accumulation in its grains and consequently targeting vitamin A deficiency. Biosynthesis of carotenoids in the endosperm of white rice is blocked at the first enzymatic step wherein geranylgeranyl diphosphate is converted to phytoene by the action of phytoene synthase (PSY). Strategies aimed at enhancing ß-carotene levels in the endosperm of white rice identified Narcissus pseudonarcissus (npPSY) and bacterial CRT1 as the regulators of the carotenoid biosynthetic pathway in rice. Besides transcriptional regulation of PSY, posttranscriptional regulation of PSY expression by OR gene, molecular synergism between ε-LCY and ß-LCY and epigenetic control of CRITSO through SET DOMAIN containing protein appear to be the other regulatory nodes which regulate carotenoid biosynthesis and accumulation in rice grains. In this review, we elucidate a comprehensive and deeper understanding of the regulatory mechanisms of carotenoid metabolism in crops that will enable us to identify an effective tool to alleviate carotenoid content in rice grains.

Transcription activation of ß-carotene biosynthetic genes at the initial stage of stresses as an indicator of the increased ß-carotene accumulation in isolated Dunaliella salina strain GY-H13.

ß-carotene is an efficient antioxidant and its accumulation is an oxidative response to stressors. Dunaliella salina strain GY-H13 is rich in ß-carotene under environmental stresses, which was selected as material to understand the molecular mechanism underlying ß-carotene biosynthesis. Seven full length cDNA sequences in ß-carotene biosynthesis pathway were cloned, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), 15-cis-zeta-carotene isomerase (ZISO), zeta-carotene desaturase (ZDS), prolycopene isomerase (CRTISO), lycopene beta-cyclase (LCYb). The seven protein sequences from the strain GY-H13 showed the highest similarity with other D. salina strains. Especially, PSY, PDS and LCYb protein sequences shared 100 % identity. Phylogenetic analysis indicated all proteins from GY-H13 firstly clustered with those from other D. salina strains with a bootstrap of 100 %. Multiple alignment indicated several distinct conserved motifs such as aspartate-rich domain (ARD), dinucleotide binding domain (DBD), and carotene binding domain (CBD). These motifs are located near ligand-binding pocket, which may be required for the activity of enzyme. Expression levels of these genes and ß-carotene content were measured over 24-h cycle, showing clear daily dynamics. All genes were dramatically up-regulated in the morning but the highest accumulation of ß-carotene was observed at noon, suggesting a lag-effect between gene transcription and biological response. Furthermore, the accumulation of ß-carotene increased under nitrogen deficiency, Cd exposure and high light and decreased under high salinity in a time-dependent manner. No gene of ß-carotene biosynthesis was up-regulated by high salinity while most genes were activated by the other stresses at the beginning stage of exposure. Growth inhibition and oxidative damage were also observed under high salinity. Overall, transcription activation of ß-carotene biosynthetic genes at the initial stage of stress exposure is a determinant of the increased accumulation of ß-carotene in microalgae, which help their survive under harsh environments. The newly isolated D. salina strain GY-H13 would be a promising microalgae model for investigating the molecular mechanism of stress-induced ß-carotene biosynthesis.

CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for ß-carotene biosynthesis in banana fruit.

Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the ß-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of ß-carotene content up to 6-fold (~24 µg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.