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The effect of salt stress on pigment synthesis and antioxidant enzyme activity as well as in the genes involved in the biosynthetic pathway of bixin was studied. The 14-day germinated seedlings of Bixa orellana were induced into the various NaCl concentration (0, 25, 50, 75, 100 mM). After 45 days, leaves were taken for pigment analysis, antioxidant assays, and gene expression analysis to study the response of salt stress. The pigment content such as chlorophyll level was increased upon salt stress with a reduction in total carotenoid clearly indicating the adaptability of plants towards the stressed state. The level of β-carotene was increased in the highest concentration of salt stress treatment. The secondary metabolites such as bixin and abscisic acid (ABA) content were also high in elevated concentration of salt-treated seedling than control. The antioxidant enzyme activity was increased with the highest dose of salt stress suggesting the antioxidant enzymes to protect the plant from the deleterious effects. The mRNA transcript gene of the carotenoid biosynthetic pathway such as phytoene synthase (PSY), 1-deoxyxylulose-5-phosphate synthase (DXS), phytoene desaturase (PDS), beta-lycopene cyclase (LCY-β), epsilon lycopene cyclase (LCY-ε), carboxyl methyl transferase (CMT), aldehyde dehydrogenase (ADH), lycopene cleavage dioxygenase (LCD), and carotenoid cleavage dioxygenase (CCD) showed differential expression pattern under salt stress. In addendum, we studied the co-expression network analysis of gene to assess the co-related genes associated in the biosynthesis pathway of carotenoid. From the co-expression analysis result showed, the LCY, PDS, and PSY genes were closely correlated with other genes. These finding may provide insight to the plants to exist in the stress condition and to improve the industrially important pigment production.

Pummelo ( Citrus maxima ) is one of important fruit trees, which belongs to Citrus species. The fruits of different pummelo cultivars have different colors and differ in the contents of carotenoid. Our results clearly showed that 'Huangjinmiyou' (HJMY) has the highest content of β-carotene, followed by 'Hongroumiyou' (HRMY) and 'Guanximiyou' (GXMY). Lycopene is dominantly accumulated in HRMY. However, the molecular mechanism underlying the carotenoid accumulation in pummelo flesh is not fully understood. In this study, we used the RNA-Seq technique to investigate the candidate genes of carotenoid metabolism in the flesh of pummelo cv. GXMY and its mutants HRMY and HJMY in three development periods of fruit. After data assembly and bioinformatic analysis, a total of 357 genes involved in biosynthesis of secondary metabolites were isolated, of which 12 differentially expressed genes (DEGs) are involved in carotenoid biosynthesis. Among these 12 DEGs, phytoene synthase ( PSY2 ), lycopene β-cyclase ( LYCB2 ), lycopene Ɛ-cyclase ( LYCE ), carotenoid cleavage dioxygenases ( CCD4 ), 9-cis-epoxycarotenoid dioxygenase ( NCED2 ), aldehyde oxidase 3 ( AAO3 ), and ABA 8'-hydroxylases ( CYP707A1 ) are the most distinct DEGs in three pummelo cultivars. The co-expression analysis revealed that the expression patterns of several transcription factors such as bHLH , MYB , ERF , NAC and WRKY are highly correlated with DEGs, which are involved in carotenoid biosynthesis. In addition, the expression patterns of 22 DEGs were validated by real-time quantitative PCR (RT-qPCR) and the results are highly concordant with the RNA-Seq results. Our results provide a global vision of transcriptomic profile among three pummelo cultivars with different pulp colors. These results would be beneficial to further study the molecular mechanism of carotenoid accumulation in pummelo flesh and help the breeding of citrus with high carotenoid content.

Rhodotorula is a group of pigment-producing yeasts well known for its intracellular biosynthesis of carotenoids such as β-carotene, γ-carotene, torulene and torularhodin. The great potential of carotenoids in applications in food and feed as well as in health products and cosmetics has generated a market value expected to reach over $2.0 billion by 2022. Due to growing public concern over food safety, the demand for natural carotenoids is rising, and this trend significantly encourages the use of microbial fermentation for natural carotenoid production. This review covers the biological properties of carotenoids and the most recent findings on the carotenoid biosynthetic pathway, as well as strategies for the metabolic engineering methods for the enhancement of carotenoid production by Rhodotorula. The practical approaches to improving carotenoid yields, which have been facilitated by advancements in strain work as well as the optimization of media and fermentation conditions, were summarized respectively.

The remarkable drought-resistance of the terrestrial cyanobacterium Nostoc flagelliforme (N. flagelliforme) has attracted attention for many years. In this study, we purified a group of red proteins that accumulate in dried field samples of N. flagelliforme. These red proteins contain canthaxanthin as the bound chromophore. Native-PAGE analysis revealed that the purified red proteins resolved into six visible red bands and were composed of four helical carotenoid proteins (HCPs), HCP1, HCP2, HCP3, and HCP6 (homologs to the N-terminal domain of the orange carotenoid protein (OCP)). Seven genes encode homologs of the OCP in the genome of N. flagelliforme: two full-length ocp genes (ocpx1 and ocpx2), four N-terminal domain hcp genes (hcp1, hcp2, hcp3, and hcp6), and one C-terminal domain ccp gene. The expression levels of hcp1, hcp2, and hcp6 were highly dependent on the water status of field N. flagelliforme samples, being downregulated during rehydration and upregulated during subsequent dehydration. Transcripts of ocpx2 were dominant in the dried field samples, which we confirmed by detecting the presence of OCPx2-derived peptides in the purified red proteins. The results shed light on the relationship between carotenoid-binding proteins and the desiccation resistance of terrestrial cyanobacteria, and the physiological functions of carotenoid-binding protein complexes in relation to desiccation are discussed.

Reptiles use pterin and carotenoid pigments to produce yellow, orange, and red colors. These conspicuous colors serve a diversity of signaling functions, but their molecular basis remains unresolved. Here, we show that the genomes of sympatric color morphs of the European common wall lizard ( Podarcis muralis ), which differ in orange and yellow pigmentation and in their ecology and behavior, are virtually undifferentiated. Genetic differences are restricted to two small regulatory regions near genes associated with pterin [ sepiapterin reductase ( SPR )] and carotenoid [ beta-carotene oxygenase 2 ( BCO2 )] metabolism, demonstrating that a core gene in the housekeeping pathway of pterin biosynthesis has been coopted for bright coloration in reptiles and indicating that these loci exert pleiotropic effects on other aspects of physiology. Pigmentation differences are explained by extremely divergent alleles, and haplotype analysis revealed abundant transspecific allele sharing with other lacertids exhibiting color polymorphisms. The evolution of these conspicuous color ornaments is the result of ancient genetic variation and cross-species hybridization., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Naturally occurring carotenoids have been isolated and used as colorants, antioxidants, nutrients, etc. in many fields. There is an ever-growing demand for carotenoids production. To comfort this, microbial production of carotenoids is an attractive alternative to current extraction from natural sources. This review summarizes the biosynthetic pathway of carotenoids and progresses in metabolic engineering of various microorganisms for carotenoid production. The advances in synthetic pathway and systems biology lead to many versatile engineering tools available to manipulate microorganisms. In this context, challenges and possible directions are also discussed to provide an insight of microbial engineering for improved production of carotenoids in the future.

β-carotene-15,15-dioxygenase is an enzyme involved in carotenoid metabolism to catalyze oxidative cleavage of β-carotene at its central double bond to two molecules of retinal in intestinal cells of vertebrate. In this study, we cloned and characterized β-carotene-15,15-dioxygenase in pearl oyster Pinctada fucata martensii (PmβCDOX). The full length of PmβCDOX gene was 1802 bp, including 1554 bp of the open reading frame (ORF) that encoded 517 amino acids, a 5'UTR of 134 bp and a 3' UTR of 114 bp. PmβCDOX was expressed at various tissues with highest level in hepatopancreas. Eighteen and fifteen single nucleotide polymorphisms (SNPs) were separately obtained in the exon and promoter of PmβCDOX. Eight SNPs (six SNPs in the exon and two SNPs in the promoter region) were significantly associated to total carotenoid content (TCC) (P < .05). The eight SNPs of significantly associated TCC were divided three haploblocks. Haplotypes CCTT had larger TCC than other haplotypes. The present results suggest that PmβCDOX is involved in carotenoid metabolism in pearl oyster. Our study will be helpful for development gene marker in selective breeding programs for TCC trait of the species., (Copyright © 2018. Published by Elsevier Inc.)

Carotenoid pigments are valuable components of the human diet. A notable example is β-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond β-carotene, we specifically used a Capsicum β-carotene hydroxylase gene, B (CaBch) and a codon optimized version of the same gene, stB (stBch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (CaBch-2A-HpBkt), consisting of the CaBch sequence and a Haematococcus β-carotene ketolase gene (HpBkt) linked by a bicistronic 2 A sequence, as well as a codon optimized recombinant stBAK gene (stBch-2A-stBkt) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (CaPsy-2A-PaCrtI) cassette to previously impart β-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8 μg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 μg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 μg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on β-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S 1 and Zea ●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S 1 and Zea ●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica ., Competing Interests: Conflict of interest statement: K.K.N. and R.E.B. are coauthors on a 2015 Perspective article.

The green microalga, Tetraselmis suecica, is commonly used in scientific, industrial, and aquacultural purposes because of its high stress tolerance and ease of culture in wide spectrums of environments. We hypothesized that carotenoids help to protect Tetraselmis cells from environmental stress by regulating genes in biosynthetic pathways. Here, we determined three major carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), and β-lycopene cyclase (LCY-B) in T. suecica, and examined the physiological parameters and gene expression responses when exposed to redox-active metals and non-redox-active metals. Phylogenetic analyses of each gene indicated that T. suecica clustered well with other green algae. Real-time PCR analysis showed that TsPSY, TsPDS, and TsLCY-B genes greatly responded to the redox-active metals in CuSO 4 followed by CuCl 2 , but not to the non-redox-active metals. The redox-active metals strongly affected the physiology of the cells, as determined by cell counting, reactive oxygen species (ROS) imaging, and photosynthetic efficiency. This suggests that carotenoids protect the cells from oxidative damage caused by metals, thereby contributing to cell survival under various stress conditions.

The red yeast Xanthophyllomyces dendrorhous was genetically engineered for high-yield accumulation of the carotenoid zeaxanthin. Initially, an astaxanthin hyper-producing mutant was used to generate a β-carotene synthesizing transformant by inactivation of the astaxanthin synthase gene. Subsequently, a bacterial β-carotene hydroxylase gene was genome integrated to establish β-carotene to zeaxanthin conversion. Crucial for efficient zeaxanthin formation was the rate of this hydroxylation which was related to the number of integrated gene copies. Two strategies were followed to get multiple integrations, either random integration into the ribosomal DNA which resulted in a maximum copy number of 10, or directly integration of a total of 8 copies into both alleles of the astaxanthin synthase gene. Combining both procedures with additional insertion of the gene to enhance expression of the carotenogenesis limiting phytoene synthase, a transformant reaching a high level of zeaxanthin of 5.2 mg/g dw was finally generated. The application of pentose sugars including xylose as substrates for X. dendrorhous which avoids the inhibitory Crab-tree effect of glucose is favorable for carotenogenesis allowing the replacement of glucose by a hydrolysate of the waste product hemicellulose which is rich in xylose demonstrating ithe effectiveness as a sustainable and cost-efficient alternative for high-yield zeaxanthin formation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microbial synthesis represents an alternative approach for the sustainable production of chemicals, fuels, and medicines. However, construction of biosynthetic pathways always suffers from side reactions, toxicity of intermediates, or low efficiency of substrate channeling. Subcellular compartmentalization may contribute to a more efficient production of target products by reducing side reactions and toxic effects within a compact insular space. The peroxisome, a type of organelle that is involved in catabolism of fatty acids and reactive oxygen species, has attracted a great deal of attention in the construction of eukaryotic cell factories with little impact on essential cellular function. In this chapter, we will systematically review recent advances in peroxisomal compartmentalization for microbial production of valuable biomolecules. Additionally, detailed experimental designs and protocols are also described. We hope a comprehensive understanding of peroxisomes will promote their application in metabolic engineering and synthetic biology., (© 2019 Elsevier Inc. All rights reserved.)

Carrots are among the richest sources of provitamin A carotenes in the human diet, but genetic variation in the carotenoid pathway does not fully explain the high levels of carotenoids in carrot roots. Using a diverse collection of modern and historic domesticated varieties, and wild carrot accessions, an association analysis for orange pigmentation revealed a significant genomic region that contains the Or gene, advancing it as a candidate for carotenoid presence in carrot. Analysis of sequence variation at the Or locus revealed a nonsynonymous mutation cosegregating with carotenoid content. This mutation was absent in all wild carrot samples and nearly fixed in all orange domesticated samples. Or has been found to control carotenoid presence in other crops but has not previously been described in carrot. Our analysis also allowed us to more completely characterize the genetic structure of carrot, showing that the Western domesticated carrot largely forms one genetic group, despite dramatic phenotypic differences among market classes. Eastern domesticated and wild accessions form a second group, which reflects the recent cultivation history of carrots in Central Asia. Other wild accessions form distinct geographic groups, particularly on the Iberian peninsula and in Northern Africa. Using genome-wide F st , nucleotide diversity, and the cross-population composite likelihood ratio, we analyzed the genome for regions putatively under selection during domestication and identified 12 regions that were significant for all three methods of detection, one of which includes the Or gene. The Or domestication allele appears to have been selected after the initial domestication of yellow carrots in the East, near the proposed center of domestication in Central Asia. The rapid fixation of the Or domestication allele in almost all orange and nonorange carrots in the West may explain why it has not been found with less genetically diverse mapping populations., (Copyright © 2018 by the Genetics Society of America.)

Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI Actinobacteria Members of this cosmopolitan lineage are proposed to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins ( actR ). This model has been difficult to validate experimentally because acI Actinobacteria are not consistently culturable. Based primarily on genomes from single cells and metagenomes, we provide a detailed biosynthetic route for members of acI clades A and B to synthesize retinal and its carotenoid precursors. Consequently, acI cells should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, unlike many bacteria that encode opsins but may need to exogenously obtain retinal because they lack retinal machinery. Moreover, we show that all acI clades contain genes for a secondary branch of the carotenoid pathway, implying synthesis of a complex carotenoid. Transcription analysis of acI Actinobacteria in a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and that actR is among the most highly transcribed of all acI genes. Furthermore, heterologous expression of acI retinal pathway genes showed that lycopene, retinal, and ActR can be made using the genes encoded in these organisms. Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed holo-ActR and acidified solution during illumination. Taken together, our results prove that acI Actinobacteria containing both ActR and acI retinal production machinery have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin. IMPORTANCE Microbes play critical roles in determining the quality of freshwater ecosystems, which are vital to human civilization. Because acI Actinobacteria are ubiquitous and abundant in freshwater lakes, clarifying their ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production, and energy cycling in freshwater habitats., (Copyright © 2018 American Society for Microbiology.)

Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes.

Novel synthetic isoprenoids have been synthesized in engineered microbial hosts by evolving terpene synthase or expressing heterologous terpene synthases. Recently, the native operon, crtN a N c M derived from Planococcus sp. PAMC 21323, has isolated for potential industrial applications of C 35 carotenoids. For the first time, novel C 35 carotenoids (sesquarterpene) were synthesized in Corynebacterium glutamicum expressing the crtN a N c M genes. The recombinant strains accumulate various sesquarterpene including 4-apolycopene (red color), 4-aponeurosporene (yellow color), and no pigmentation, depending on the expression of the genetic elements of the crtN a N c M genes. Subsequently, the carotenoid extract from the cells harboring pCES-H36-CrtN a N c M was analyzed, resulting in significantly higher antioxidant activity than those of other strains harboring pCES-H36-CrtN c M and pCES-H36-CrtN a N c , respectively. This study will promote further engineering of C. glutamicum to increase sesquarterpene productions.

Light-emitting diodes (LEDs) are considered the future of greenhouse lighting. This study investigates the carotenoid concentrations of pak choi sprouts after growth under blue, red and white LEDs at six different time points. Furthermore, the diurnal changes of RNA transcripts of key genes of the carotenoid biosynthesis pathway as well as of the carotenoid cleavage dioxygenase 4 (CCD4) gene and of the transcription factor genes elongated hypocotyl 5 (HY5) and circadian clock associated 1 (CCA1) were investigated. The carotenoid concentrations were steady throughout the day, but showed a small maximum in the afternoon. An average total carotenoid concentration of 536 ± 29 ng mg-1 DM produced under white LEDs was measured, which is comparable to previously described field-grown levels. The carotenoid concentrations were slightly lower under blue or red LEDs. Moreover, the diurnal RNA transcript rhythms of most of the carotenoid biosynthesis genes showed an increase during the light period, which can be correlated to the carotenoid maxima in the afternoon. Blue LEDs caused the highest transcriptional induction of biosynthetic genes as well as of CCD4, thereby indicating an increased flux through the pathway. In addition, the highest levels of HY5 transcripts and CCA1 transcripts were determined under blue LEDs.

Chlorobaculum tepidum , a green sulfur bacterium, utilizes chlorobactene as its major carotenoid, and this organism also accumulates a reduced form of this monocyclic pigment, 1',2'-dihydrochlorobactene. The protein catalyzing this reduction is the last unidentified enzyme in the biosynthetic pathways for all of the green sulfur bacterial pigments used for photosynthesis. The genome of C. tepidum contains two paralogous genes encoding members of the FixC family of flavoproteins: bchP , which has been shown to encode an enzyme of bacteriochlorophyll biosynthesis; and bchO , for which a function has not been assigned. Here we demonstrate that a bchO mutant is unable to synthesize 1',2'-dihydrochlorobactene, and when bchO is heterologously expressed in a neurosporene-producing mutant of the purple bacterium, Rhodobacter sphaeroides , the encoded protein is able to catalyze the formation of 1,2-dihydroneurosporene, the major carotenoid of the only other organism reported to synthesize 1,2-dihydrocarotenoids, Blastochloris viridis Identification of this enzyme completes the pathways for the synthesis of photosynthetic pigments in Chlorobiaceae , and accordingly and consistent with its role in carotenoid biosynthesis, we propose to rename the gene cruI Notably, the absence of cruI in B. viridis indicates that a second 1,2-carotenoid reductase, which is structurally unrelated to CruI (BchO), must exist in nature. The evolution of this carotenoid reductase in green sulfur bacteria is discussed herein., (© 2018 Canniffe et al.)

Background: An Immunochip study recently identified the association of a number of new genetic loci with Behcet's disease (BD)., Objective: To confirm the association between new genetic loci reported in an Immunochip study and BD in a Han Chinese population., Methods: A two-stage association study was carried out in 1238 patients with BD and 1458 healthy controls. Twenty-two candidate single nucleotide polymorphisms (SNPs) were selected for genotyping by iPLEXGold genotyping or TaqMan SNP assays and a meta-analysis was performed for significantly associated markers., Results: The results showed that four SNPs ( LACC1 /rs9316059, CEBPB-PTPN1 /rs913678, ADO-EGR2 /rs224127 and RIPK2 /rs10094579) were associated with BD in an allelic association test (rs9316059 T allele: p c =4.95×10 -8 , OR=0.687; rs913678 C allele: p c =3.01×10 -4 , OR=1.297; rs224127 A allele: p c =3.77×10 -4 , OR=1.274; rs10094579 A allele: p c =6.93×10 -4 , OR=1.302). For four SNPs tested by meta-analysis, the association with BD was strengthened and all exceeded genome-wide significance (rs9316059: p=2.96×10 -16 ; rs913678: p=2.09×10 -16 ; rs224127: p=5.28×10 -13 ; rs10094579: p=9.21×10 -11 )., Conclusions: Our findings confirmed the association of four loci ( LACC1 , CEBPB-PTPN1 , ADO-EGR2 and RIPK2 ) in Chinese Han patients with BD., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4'-diapophytofluene and 4,4'-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1-3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4'-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4'-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4'-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Carrot (Daucus carota L.) is one of the most economically important root vegetables in the world, providing numerous nutrients for human health. China is the largest country of carrot production in the world, and 'Kurodagosun' has been a major carrot variety in China. Carrot material used in this study was the inbred line 'DC-27', which was derived by forced selfing from 'Kurodagosun'. To understand the genetic system and plant-specific genes of 'Kurodagosun', we report the draft genome sequence of carrot 'DC-27' assembled using a combination of Roche454 and HiSeq 2000 sequencing technologies to achieve 32-fold genome coverage. A total of 31,891 predicted genes were identified. These assembled sequences provide candidate genes involved in biological processes including stress response and carotenoid biosynthesis. Genomic sequences corresponding to 371.6 Mb was less than 473 Mb, which is the estimated genome size. The availability of a draft sequence of the 'DC-27' genome advances knowledge on the biological research and breeding of carrot, as well as other Apiaceae plants. The 'DC-27' genome sequence data also provide a new resource to explore the evolution of other higher plants.

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.

Angiosperms germinated in the dark develop etioplasts, the chloroplast precursors, in cotyledon cells. Etioplasts contain lattice membrane structures called prolamellar bodies (PLBs) and lamellar prothylakoids as internal membrane systems. PLBs accumulate the chlorophyll intermediate protochlorophyllide (Pchlide) in a complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). Two galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG), are major constituents of etioplast membranes. We previously reported that monogalactosyldiacylglycerol facilitates the synthesis of Pchlide and the formation of the Pchlide-LPOR-NADPH complex in etioplasts, but the importance of DGDG in etioplasts is still unknown. To determine the role of DGDG in etioplast development and functions, we characterized a knockout mutant ( dgd1 ) of Arabidopsis ( Arabidopsis thaliana ) DGD1 , which encodes the major isoform of DGDG synthase, in the etioplast development stage. In etiolated dgd1 seedlings, DGDG content decreased to 20% of the wild-type level, the lattice structure of PLBs was disordered, and the development of prothylakoids was impaired. In addition, membrane-associated processes of Pchlide biosynthesis, formation of the Pchlide-LPOR-NADPH complex, and dissociation of the complex after the photoconversion of Pchlide to chlorophyllide were impaired in dgd1 , although the photoconversion reaction by LPOR was not affected by the DGDG deficiency. Total carotenoid content also decreased in etiolated dgd1 seedlings, but the carotenoid composition was unchanged. Our data demonstrate a deep involvement of DGDG in the formation of the internal membrane structures in etioplasts as well as in membrane-associated processes of pigment biosynthesis and pigment-protein complex organization., (© 2018 American Society of Plant Biologists. All rights reserved.)

Carotenoids in citrus fruits have health benefits and make the fruits visually attractive. Red-fleshed 'Chuhong' ('CH') and pale green-fleshed 'Feicui' ('FC') pummelo (Citrus maxima (Burm) Merr.) fruits are interesting materials for studying the mechanisms of carotenoid accumulation. In this study, particularly high contents of linear carotenes were observed in the albedo tissue, segment membranes and juice sacs of 'CH'. However, carotenoids, especially β-carotene and xanthophylls, accumulated more in the flavedo tissue of 'FC' than in that of 'CH'. Additionally, the contents of other terpenoids such as limonin, nomilin and abscisic acid significantly differed in the juice sacs at 150 days postanthesis. A dramatic increase in carotenoid production was observed at 45 to 75 days postanthesis in the segment membranes and juice sacs of 'CH'. Different expression levels of carotenogenesis genes, especially the ζ-carotene desaturase (CmZDS), β-carotenoid hydroxylase (CmBCH) and zeaxanthin epoxidase (CmZEP) genes, in combination are directly responsible for the largely different carotenoid profiles between these two pummelo fruits. The sequences of eleven genes involved in carotenoid synthesis were investigated; different alleles of seven of eleven genes might also explain the largely different carotenogenesis observed between 'CH' and 'FC'. These results enhance our understanding of carotenogenesis in pummelo fruits., Competing Interests: The authors have declared that no competing interests exist.

Annatto (Bixa orellana L.) is a tropical American crop, commercially valuable due to its application in the food and cosmetics industries as a natural dye. The wild ancestor of cultivated annatto is B. orellana var. urucurana. Although never cultivated, this variety occurs in open forests and anthropogenic landscapes, and is always associated with riparian environments. In this study, we evaluated the genetic diversity and structure of B. orellana var. urucurana populations in Brazilian Amazonia using 16 microsatellite loci. We used Ecological Niche Modeling (ENM) to characterize the potential geographical range of this variety in northern South America. We analyzed 170 samples from 10 municipalities in the states of Rondônia, Pará and Roraima. A total of 194 alleles was observed, with an average of 12.1 alleles per locus. Higher levels of expected (HE) than observed (HO) heterozygosities were found for all populations. Bayesian analysis, Neighbor-Joining dendrograms and PCAs suggest the existence of three strongly structured groups of populations. A strong and positive correlation between genetic and geographic distances was found, suggesting that genetic differentiation might be caused by geographic isolation. From species distribution modelling, we detected that South Rondônia, Madre di Dios River basin, Llanos de Mojos, Llanos de Orinoco and eastern Ecuador are highly suitable areas for wild annatto to occur, providing additional targets for future exploration and conservation. Climatic adaptation analyses revealed strong differentiation among populations, suggesting that precipitation plays a key role in wild annatto's current and potential distribution patterns., Competing Interests: The authors have declared that no competing interests exist.

Carotenoids are a diverse group of tetraterpenoid pigments that play indispensable roles in plants and animals. The biosynthesis of carotenoids in plants is strictly regulated at the transcriptional and post-transcriptional levels in accordance with inherited genetic signals and developmental requirements and in response to external environmental stimulants. The alteration in the biosynthesis of carotenoids under the influence of external environmental stimulants, such as high light, drought, salinity, and chilling stresses, has been shown to significantly influence the nutritional value of crop plants. In addition to these stimulants, several pre- and postharvesting cultivation practices significantly influence carotenoid compositions and contents. Thus, this review discusses how various environmental stimulants and pre- and postharvesting factors can be positively modulated for the enhanced biosynthesis and accumulation of carotenoids in the edible parts of crop plants, such as the leaves, roots, tubers, flowers, fruit, and seeds. In addition, future research directions in this context are identified.

A targeted approach using HS-SPME-GC-MS was performed to investigate volatile compounds of ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV) at different developmental stages. Fifty-six volatile components classified into six chemical groups were quantified. The individual volatile compounds in each group were significantly influenced by the genotype and harvest date. Cluster analysis suggested two principal clusters. Cluster I included fruits of VAL and RRV harvested in July, September, and November and was characterized by the 2-ethyl-1-hexanol acetate and linalool chemotype; cluster II included fruits harvested in January and March, with the valencene/β-myrcene chemotype. Principal component analysis confirmed that fruits of both cultivars harvested at different dates possessed a unique aroma active profile, especially, the odor-active volatile-norisoprenoids degraded from C 40 carotenoid were significantly higher in RRV than in VAL at full maturity. These results could provide information for further study of the flavor metabolism pathways in the two closely related cultivars., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Anther cuticle and pollen exine are two elaborated lipid-soluble barriers protecting pollen grains from environmental and biological stresses. However, less is known about the mechanisms underlying the synthesis of these lipidic polymers. Here, we identified a no-pollen male-sterility mutant cyp703a3-3 from the indica restorer line Zhonghui 8015 (Zh8015) mutant library treated with 60 Coγ-ray radiation. Histological analysis indicated that cyp703a3-3 underwent abnormal tapetal cells development, produced few orbicules and secreted less sporopollenin precursors to anther locule, as well as cutin monomers on anther. Genetic analysis revealed that cyp703a3-3 was controlled by a single recessive gene. Map-based cloning was performed to narrow down the mutant gene to a 47.78-kb interval on the chromosome 8 between two markers S15-29 and S15-30. Sequence analysis detected three bases (GAA) deletion in the first exon of LOC&#95;Os08g03682, annotated as CYP703A3 with homologous sequences related to male sterility in Arabidopsis, causing the Asparagine deletion in the mutant site. Moreover, we transformed genomic fragment of CYP703A3 into cyp703a3-3, which male-sterility phenotype was recovered. Both the wild-type and cyp703a3-3 mutant 3D structure of CYP703A3 protein were modeled. Results of qPCR suggested CYP703A3 mainly expressed in anthers with greatest abundance at microspore stage, and genes involved in sporopollenin precursors formation and transportation, such as GAMYB, TDR, CYP704B2, DPW2, OsABCG26 and OsABCG15, were significantly reduced in cyp703a3-3. Collectively, our results further elaborated CYP703A3 plays vital role in anther cuticle and pollen exine development in rice (Oryza sativa L.)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The lycopene biosynthetic genes crtE, crtB, and crtI from Deinococcus wulumuqiensis R12 were integrated into three different vector backbones-pET28a, pTrc99A, and pUC18-and the resulting recombinant plasmids pET28a-EBI, pTrc99A-EBI, and pUC18-EBI were introduced into different Escherichia coli hosts. The results showed that lycopene production of strain 28BL was lower than that of 99 series strains without IPTG in LB medium. In addition, lycopene production of 99JM with supplementation of 20% (w/v) glycerol was 1.6-fold higher than with supplementation of 6% (w/v) glucose. After optimization of the host strain and culture medium, the yield of microbial lycopene was increased successfully. When recombinant E. coli 99DH was cultivated under exposure to light in 2YT + Gly medium, the highest lycopene production rate was 26.2 mg/L/h at 30 h, and the maximum specific lycopene content was 67 mg/g dry cell (925 mg/L) at 40 h, which represents a 76% increase over the starting point.

Permafrost accounts for 27% of all soil ecosystems and harbors diverse microbial communities. Our understanding of microorganisms in permafrost, their activities and adaptations, remains limited. Using five subzero-growing (cryophilic) permafrost bacteria, we examined features of cold adaptation through comparative genomic analyses with mesophilic relatives. The cryophiles possess genes associated with cold adaptation, including cold shock proteins, RNA helicases, and oxidative stress and carotenoid synthesis enzymes. Higher abundances of genes associated with compatible solutes were observed, important for osmoregulation in permafrost brine veins. Most cryophiles in our study have higher transposase copy numbers than mesophiles. We investigated amino acid (AA) modifications in the cryophiles favoring increased protein flexibility at cold temperatures. Although overall there were few differences with the mesophiles, we found evidence of cold adaptation, with significant differences in proline, serine, glycine and aromaticity, in several cryophiles. The use of cold/hot AA ratios of >1, used in previous studies to indicate cold adaptation, was found to be inadequate on its own. Comparing the average of all cryophiles to all mesophiles, we found that overall cryophiles had a higher ratio of cold adapted proteins for serine (more serine), and to a lesser extent, proline and acidic residues (fewer prolines/acidic residues).

The order Sphingomonadales is a taxon of bacteria with a variety of physiological features and carotenoid pigments. Some of the coloured strains within this order are known to be aerobic anoxygenic phototrophs that contain characteristic photosynthesis gene clusters (PGCs). Previous work has shown that majority of the ORFs putatively involved in the biosynthesis of C40 carotenoids are located outside the PGCs in these strains. The main purpose of this study was to understand the genetic basis for the various colour/carotenoid phenotypes of the strains of Sphingomonadales. Comparative analyses of the genomes of 41 strains of this order revealed that there were different patterns of clustering of carotenoid biosynthesis (crt) ORFs, with four ORF clusters being the most common. The analyses also revealed that co-occurrence of crtY and crtI is an evolutionarily conserved feature in Sphingomonadales and other carotenogenic bacteria. The comparisons facilitated the categorisation of bacteria of this order into four groups based on the presence of different crt ORFs. Yellow coloured strains most likely accumulate nostoxanthin, and contain six ORFs (group I: crtE, crtB, crtI, crtY, crtZ, crtG). Orange coloured strains may produce adonixanthin, astaxanthin, canthaxanthin and erythroxanthin, and contain seven ORFs (group II: crtE, crtB, crtI, crtY, crtZ, crtG, crtW). Red coloured strains may accumulate astaxanthin, and contain six ORFs (group III: crtE, crtB, crtI, crtY, crtZ, crtW). Non-pigmented strains may contain a smaller subset of crt ORFs, and thus fail to produce any carotenoids (group IV). The functions of many of these ORFs remain to be characterised.

The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides ) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi., Competing Interests: SC, DP, GG, MI, SM, MR, YC, SB, IG, JG, BS, RB, AA, JS No competing interests declared

Maize white seedling 3 (w3) has been used to study carotenoid deficiency for almost 100 years, although the molecular basis of the mutation has remained unknown. Here we show that the w3 phenotype is caused by disruption of the maize gene for homogentisate solanesyl transferase (HST), which catalyzes the first and committed step in plastoquinone-9 (PQ-9) biosynthesis in the plastid. The resulting PQ-9 deficiency prohibits photosynthetic electron transfer and eliminates PQ-9 as an oxidant in the enzymatic desaturation of phytoene during carotenoid synthesis. As a result, light-grown w3 seedlings are albino, deficient in colored carotenoids and accumulate high levels of phytoene. However, despite the absence of PQ-9 for phytoene desaturation, dark-grown w3 seedlings can produce abscisic acid (ABA) and homozygous w3 kernels accumulate sufficient carotenoids to generate ABA needed for seed maturation. The presence of ABA and low levels of carotenoids in w3 nulls indicates that phytoene desaturase is able to use an alternate oxidant cofactor, albeit less efficiently than PQ-9. The observation that tocopherols and tocotrienols are modestly affected in w3 embryos and unaffected in w3 endosperm indicates that, unlike leaves, grain tissues deficient in PQ-9 are not subject to severe photo-oxidative stress. In addition to identifying the molecular basis for the maize w3 mutant, we: (1) show that low levels of phytoene desaturation can occur in w3 seedlings in the absence of PQ-9; and (2) demonstrate that PQ-9 and carotenoids are not required for vitamin E accumulation., (© 2018 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

The MADS-box transcription factors play essential roles in many physiological and biochemical processes of plants, especially in fruit ripening. Here, a tomato MADS-box gene, SlCMB1, was isolated. SlCMB1 expression declined with the fruit ripening from immature green to B + 7 (7 days after Breaker) fruits in the wild type (WT) and was lower in Nr and rin mutants fruits. Tomato plants with reduced SlCMB1 mRNA displayed delayed fruit ripening, reduced ethylene production and carotenoid accumulation. The ethylene production in SlCMB1-RNAi fruits decreased by approximately 50% as compared to WT. The transcripts of ethylene biosynthesis genes (ACS2, ACS4, ACO1 and ACO3), ethylene-responsive genes (E4, E8 and ERF1) and fruit ripening-related genes (RIN, TAGL1, FUL1, FUL2, LoxC and PE) were inhibited in SlCMB1-RNAi fruits. The carotenoid accumulation was decreased and two carotenoid synthesis-related genes (PSY1 and PDS) were down-regulated while three lycopene cyclase genes (CYCB, LCYB and LCYE) were up-regulated in transgenic fruits. Furthermore, yeast two-hybrid assay showed that SlCMB1 could interact with SlMADS-RIN, SlMADS1, SlAP2a and TAGL1, respectively. Collectively, these results indicate that SlCMB1 is a new component to the current model of regulatory network that regulates ethylene biosynthesis and carotenoid accumulation during fruit ripening.

Background: Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host., Results: We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence., Conclusions: T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.

Carotenoid cleavage oxygenases (CCOs) are able to cleave carotenoids to produce apocarotenoids and their derivatives, which are important for plant growth and development. In this study, 21 apple CCO genes were identified and divided into six groups based on their phylogenetic relationships. We further characterized the apple CCO genes in terms of chromosomal distribution, structure and the presence of cis-elements in the promoter. We also predicted the cellular localization of the encoded proteins. An analysis of the synteny within the apple genome revealed that tandem, segmental, and whole-genome duplication events likely contributed to the expansion of the apple carotenoid oxygenase gene family. An additional integrated synteny analysis identified orthologous carotenoid oxygenase genes between apple and Arabidopsis thaliana, which served as references for the functional analysis of the apple CCO genes. The net photosynthetic rate, transpiration rate, and stomatal conductance of leaves decreased, while leaf stomatal density increased under drought and saline conditions. Tissue-specific gene expression analyses revealed diverse spatiotemporal expression patterns. Finally, hormone and abiotic stress treatments indicated that many apple CCO genes are responsive to various phytohormones as well as drought and salinity stresses. The genome-wide identification of apple CCO genes and the analyses of their expression patterns described herein may provide a solid foundation for future studies examining the regulation and functions of this gene family., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.

A novel type strain, Planococcus faecalis AJ003 T , isolated from the feces of Antarctic penguins, synthesizes a rare C30 carotenoid, glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The complete genome of P. faecalis AJ003 T comprises a single circular chromosome (3,495,892 bp; 40.9% G + C content). Annotation analysis has revealed 3511 coding DNA sequences and 99 RNAs; seven genes associated with the MEP pathway and five genes involved in the carotenoid pathway have been identified. The functionality and complementation of 4,4'-diapophytoene synthase (CrtM) and two copies of heterologous 4,4'-diapophytoene desaturase (CrtN) involved in carotenoid biosynthesis were analyzed in Escherichia coli., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Carotenoids are isoprenoid pigments synthesized exclusively by plants and microorganisms and play critical roles in light harvesting, photoprotection, attracting pollinators and phytohormone production. In recent years, carotenoids have been used for their health benefits due to their high antioxidant activity and are extensively utilized in food, pharmaceutical, and nutraceutical industries. Regulation of carotenoid biosynthesis occurs throughout the life cycle of plants, with vibrant changes in composition based on developmental needs and responses to external environmental stimuli. With advancements in metabolic engineering techniques, there has been tremendous progress in the production of industrially valuable secondary metabolites such as carotenoids. Application of metabolic engineering and synthetic biology has become essential for the successful and improved production of carotenoids. Synthetic biology is an emerging discipline; metabolic engineering approaches may provide insights into novel ideas for biosynthetic pathways. In this review, we discuss the current knowledge on carotenoid biosynthetic pathways and genetic engineering of carotenoids to improve their nutritional value. In addition, we investigated synthetic biological approaches for the production of carotenoids. Theoretical biology approaches that may aid in understanding the biological sciences are discussed in this review. A combination of theoretical knowledge and experimental strategies may improve the production of industrially relevant secondary metabolites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

Red carotenoid colours in birds are widely assumed to be sexually selected quality indicators, but this rests on a very incomplete understanding of genetic mechanisms and honesty-mediating costs. Recent progress was made by the implication of the gene CYP2J19 as an avian carotenoid ketolase, catalysing the synthesis of red C4-ketocarotenoids from yellow dietary precursors, and potentially a major mechanism behind red coloration in birds. Here, we investigate the role of CYP2J19 in the spectacular colour diversification of African weaverbirds (Ploceidae), represented by five genera and 16 species: eight red, seven yellow and one without carotenoid coloration. All species had a single copy of CYP2J19, unlike the duplication found in the zebra finch, with high expression in the retina, confirming its function in colouring red oil droplets. Expression was weak or undetected in skin and follicles of pigment-depositing feather buds, as well as in beaks and tarsi, including those of the red-billed quelea. In contrast, the hepatic (liver) expression of CYP2J19 was consistently higher (>14-fold) in seven species with C4-ketocarotenoid coloration than in species without (including one red species), an association strongly supported by a phylogenetic comparative analysis. The results suggest a critical role of the candidate ketolase, CYP2J19, in the evolution of red C4-ketocarotenoid colour variation in ploceids. As ancestral state reconstruction suggests that ketocarotenoid coloration has evolved twice in this group (once in Euplectes and once in the Quelea/Foudia clade), we argue that while CYP2J19 has retained its ancestral role in the retina, it has likely been co-opted for red coloration independently in the two lineages, via increased hepatic expression., (© 2017 John Wiley & Sons Ltd.)

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

The color of tomato ( Solanum lycopersicum ) fruit flesh is often used as an indicator of quality. Generally, fruit color is determined by the accumulation of carotenoids and flavonoids, along with concomitant degradation of chlorophylls during ripening. Several genes, such as phytoenesynthetase1 ( Psy1 ), STAY-GREEN ( SGR ), and SlMYB12 , have been extensively studied to elucidate the genes controlling fruit coloration. In this study, we observed low carotenoid levels without degradation of chlorophylls in green-fruited tomato caused by mutations in three genes, Psy1 , SGR , and SlMYB12 . We crossed two inbred lines, BUC30 (green-fruited) and KNR3 (red-fruited), to confirm the causal effects of these mutations on fruit coloration. The F₂ population segregated for eight different fruit colors in the proportions expected for three pairs of gene, as confirmed by a chi-square test. Therefore, we developed a population of tomato with diverse fruit colors and used molecular markers to detect the genes responsible for the individual fruit colors. These newly-designed DNA-based markers can be used for selecting desired fruit color genotypes within adapted breeding materials and cultivars for breeding., Competing Interests: The authors declare no conflict of interest.

Papaya is an important tropical fruit with a rich source of carotenoids. The ripening of papaya is a physiological and metabolic process with remarkable changes including accumulation of carotenoids, which depends primarily on the action of ethylene. Ethylene response is mediated by a transcriptional cascade involving the transcription factor families of EIN3/EILs and ERFs. Although ERF members have been reported to control carotenoid production in Arabidopsis and tomato, whether EIN3/EILs are also involved in carotenoid biosynthesis during fruit ripening remains unclear. In this work, two EIN3 genes from papaya fruit, namely CpEIN3a and CpEIN3b, were studied, of which CpEIN3a was increased during fruit ripening, concomitant with the increase of transcripts of carotenoid biosynthesis-related genes including CpPDS2/4, CpZDS, CpLCY-e and CpCHY-b, and carotenoid content. Electrophoretic mobility shift assays (EMSAs) and transient expression analyses revealed that CpEIN3a was able to bind to the promoters of CpPDS4 and CpCHY-b, and promoted their transcription. Protein-protein interaction assays indicated that CpEIN3a physically interacted with another transcription factor CpNAC2, which acted as a transcriptional activator of CpPDS2/4, CpZDS, CpLCY-e and CpCHY-b by directly binding to their promoters. More importantly, the transcriptional activation abilities of CpPDS2/4, CpLCY-e and CpCHY-b were more pronounced following their interaction. Collectively, our findings suggest that CpEIN3a interacts with CpNAC2 and, individually or co-operatively, activates the transcription of a subset of carotenoid biosynthesis-related genes, providing new insights into the regulatory networks of carotenoid biosynthesis during papaya fruit ripening., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Developing computational tools for a chassis-centered biosynthetic pathway design is very important for a productive heterologous biosynthesis system by considering enormous foreign biosynthetic reactions. For many cases, a pathway to produce a target molecule consists of both native and heterologous reactions when utilizing a microbial organism as the host organism. Due to tens of thousands of biosynthetic reactions existing in nature, it is not trivial to identify which could be served as heterologous ones to produce the target molecule in a specific organism. In the present work, we integrate more than 10,000 E. coli non-native reactions and utilize a probability-based algorithm to search pathways. Moreover, we built a user-friendly Web server named EcoSynther. It is able to explore the precursors and heterologous reactions needed to produce a target molecule in Escherichia coli K12 MG1655 and then applies flux balance analysis to calculate theoretical yields of each candidate pathway. Compared with other chassis-centered biosynthetic pathway design tools, EcoSynther has two unique features: (1) allow for automatic search without knowing a precursor in E. coli and (2) evaluate the candidate pathways under constraints from E. coli physiological states and growth conditions. EcoSynther is available at http://www.rxnfinder.org/ecosynther/ .

A conventional breeding program was established to transfer the bacterial phytoene synthase transgene-crtB-from a transgenic, white-rooted cassava to yellow-rooted cassava plants carrying the endogenous phytoene synthase alleles named psy2-y 1 and/or psy2-y 2 . Combining endogenous phytoene synthase enzymes (PSYs) with CRTB in a single cassava plant would allow the molecular dissection of individual allele contributions to carotenoid synthesis and/or accumulation in cassava roots. The simultaneous expression of the crtB transgene and psy2-y 2 in individuals planted in the field coincided with higher total, HPLC-quantified carotenoid content in roots, although the variability among replications (plants) precluded the detection of statistically significant differences. Nevertheless, the highest total carotenoid content in roots within a family coincided with one individual of the F1 progeny carrying both psy2-y 2 and crtB genes. The results also indicated the presence of at least one more key gene-different from psy or crtB-which too is necessary for the synthesis and/or accumulation of Pro-Vitamin A carotenoids in cassava roots.

High-carotenoid (HC) maize, a biofortified staple crop which accumulates β-carotene, β-cryptoxanthin, lutein and zeaxanthin, was used as a feed component in a chicken feeding trial to assess the bioavailability of provitamin A (PVA) carotenoids in the kernel matrix compared to the synthetic and natural color additives routinely used in the poultry industry. We found that the PVA carotenoids in HC maize were not metabolized in the same manner: β-carotene was preferentially converted into retinol in the intestine whereas β-cryptoxanthin accumulated in the liver. We also considered the effect of zeaxanthin on the absorption of PVA carotenoids because zeaxanthin is the major carotenoid component of HC maize. We found that chickens fed on diets with low levels of zeaxanthin accumulated higher levels of retinol in the liver, suggesting that zeaxanthin might interfere with the absorption of β-carotene, although this observation was not statistically significant. Our results show that HC maize provides bioavailable carotenoids, including PVA carotenoids, and is suitable for use as a feed component.

We investigated the transcriptional regulation of six genes involved in carotenoid biosynthesis, together with the carotenoid accumulation during postharvest ripening of three different papaya genotypes of contrasting pulp color. Red-pulp genotype (RPG) showed the lowest content of yellow pigments (YP), such as β-cryptoxanthin, zeaxanthin, and violaxanthin, together with the lowest relative expression levels (REL) of CpLCY-β2 and CpCHX-β genes. On the contrary, the yellow-pulp genotype (YPG) showed the highest content of YP and the highest REL of CpLCY-β2 and CpCHX-β genes. Interestingly, the orange-pulp genotype (OPG) showed intermediate content of YP and intermediate REL of CpLCY-β2 and CpCHX-β genes. The highest content of β-carotene shown by OPG despite having an intermediate REL of the CpLCY-β2 genes, suggests a post-transcriptional regulation. Thus, the transcriptional level of the genes, directing the carotenoid biosynthesis pathway, can partially explain the accumulation of carotenoids during the postharvest ripening in C. papaya genotypes of contrasting pulp color., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Traditional varieties constitute a wide source of genetic variation that can be used per se or to obtain new cultivars with high consumer appeal and nutritional value. This work focuses on the carotenoid and chlorophyll profiles and contents of 53 traditional tomato cultivars, paying particular attention to compounds with recognized health-promoting properties. The study includes fruits with different shapes (oblate, slightly flattened, rounded, heart-shaped, long oblong and pyriform), colors (yellow, pink and red) and sizes (very small to very large). In addition, black colored tomato fruits with yellow, pink or red background color were studied. The highest concentrations of lycopene, β-carotene, phytoene and phytofluene were found in pink and red tomatoes, while the highest concentrations of lutein, violaxanthin, neoxanthin and chlorophylls were found in fruits with a dark coloration, regardless of their background coloration. Finally, the highest concentrations of the studied compounds as a whole (except β-carotene) were found in red- and pink-black varieties. Findings will hopefully contribute to recovering many tomato traditional varieties for use, directly in the field or as donor parents for breeding programs, to increase the nutraceutical properties of commercial varieties., (Copyright © 2016. Published by Elsevier Ltd.)

Pigmented metabolites have great potential for use in biosensors that target low-resource areas, since sensor output can be interpreted without any equipment. However, full repression of pigment production when undesired is challenging, as even small amounts of enzyme can catalyze the production of large, visible amounts of pigment. The red pigment lycopene could be particularly useful because of its position in the multi-pigment carotenoid pathway, but commonly used inducible promoter systems cannot repress lycopene production. In this paper, we designed a system that could fully repress lycopene production in the absence of an inducer and produce visible lycopene within two hours of induction. We engineered Lac, Ara, and T7 systems to be up to 10 times more repressible, but these improved systems could still not fully repress lycopene. Translational modifications proved much more effective in controlling lycopene. By decreasing the strength of the ribosomal binding sites on the crtEBI genes, we enabled full repression of lycopene and production of visible lycopene in 3-4h of induction. Finally, we added the mevalonate pathway enzymes to increase the rate of lycopene production upon induction and demonstrated that supplementation of metabolic precursors could decrease the time to coloration to about 1.5h. In total, this represents over an order of magnitude reduction in response time compared to the previously reported strategy. The approaches used here demonstrate the disconnect between fluorescent and metabolite reporters, help enable the use of lycopene as a reporter, and are likely generalizable to other systems that require precise control of metabolite production., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)