Hierarchical dynamic regulation of Saccharomyces cerevisiae for enhanced lutein biosynthesis.

Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.

CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase for Efficient Astaxanthin Production in the Green Microalga Chlamydomonas reinhardtii.

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

5'-UTR allelic variants and expression of the lycopene-ɛ-cyclase LCYE gene in maize (Zea mays L.) inbred lines of Russian selection.

In breeding, biofortification is aimed at enriching the edible parts of the plant with micronutrients. Within the framework of this strategy, molecular screening of collections of various crops makes it possible to determine allelic variants of genes, new alleles, and the linkage of allelic variants with morphophysiological traits. The maize (Zea mays L.) is an important cereal and silage crop, as well as a source of the main precursor of vitamin A - ß-carotene, a derivative of the ß,ß-branch of the carotenoid biosynthesis pathway. The parallel ß,ε-branch is triggered by lycopene-ε-cyclase LCYE, a low expression of which leads to an increase in provitamin A content and is associated with the variability of the 5'-UTR gene regulatory sequence. In this study, we screened a collection of 165 maize inbred lines of Russian selection for 5'- UTR LCYE allelic variants, as well as searched for the dependence of LCYE expression levels on the 5'-UTR allelic variant in the leaves of 14 collection lines. 165 lines analyzed were divided into three groups carrying alleles A2 (64 lines), A5 (31) and A6 (70), respectively. Compared to A2, allele A5 contained two deletions (at positions -267- 260 and -296-290 from the ATG codon) and a G251→T substitution, while allele A6 contained one deletion (-290-296) and two SNPs (G251→T, G265→T). Analysis of LCYE expression in the leaf tissue of seedlings from accessions of 14 lines differing in allelic variants showed no associations of the 5'-UTR LCYE allele type with the level of gene expression. Four lines carrying alleles A2 (6178-1, 6709-2, 2289-3) and A5 (5677) had a significantly higher level of LCYE gene expression (~0.018-0.037) than the other 10 analyzed lines (~0.0001-0.004), among which all three allelic variants were present.

Transcript profiling of genes involved in carotenoid biosynthesis among three carrot cultivars with various taproot colors.

Carotenoids are a group of natural pigments that are widely distributed in various plants. Carrots are plants rich in carotenoids and have fleshy roots with different colors. Carotenoid accumulation is a complex regulatory process with important guiding significance for carrot production. In this work, three carrot cultivars with different taproot colors, Hongxinqicun (orange), Benhongjinshi (red), and Tianzi (purple) were chosen as experimental materials to explore the molecular mechanism of carotenoid accumulation in carrot. Results showed that the three carotenoids, namely, α-carotene, ß-carotene, and lutein, had accumulated in orange carrot cultivar Hongxinqicun. Lycopene was only detected in the taproots of Benhongjinshi. Lutein was the main carotenoid in Tianzi. Comparison of the carotenoid contents in different tissues of carrot showed that leaf blade was the tissue with the highest carotenoid accumulation. Expression analysis of carotenoid biosynthesis genes and its correlation with carotenoid accumulation confirmed the regulatory role of structural genes in carrots. The high expression of five lycopene synthesis-related genes, DcPSY2, DcPDS, DcZDS1, DcCRT1, DcCRT2, and low expression of DcLCYE may result in the lycopene accumulation in Benhongjinshi. However, the function of certain genes, such as DcPSY1 that was lowly expressed in red carrot, requires further investigation. Our results provided potential insights into the mechanism of carotenoid accumulation in three carrot cultivars with different taproot colors.

Down-regulation of lycopene ε-cyclase expression in transgenic sweetpotato plants increases the carotenoid content and tolerance to abiotic stress.

Carotenoids are required for many biological processes in plants and humans. Lycopene ε-cyclase (LCY-ε) catalyzes the conversion of lycopene into lutein via the α-branch carotenoid biosynthesis pathway. Down-regulation of IbLCY-ε by RNAi increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato calli. As the role of IbLCY-ε in carotenoid biosynthesis and environmental stress responses in whole plants is poorly understood, transgenic sweetpotato (RLE plants) with reduced expression of IbLCY-ε were developed. RLE plants contained higher levels of total carotenoid and ß-carotene, due to an elevated ß-carotene/lutein ratio rather than increased de novo biosynthesis. RLE plants showed high reactive oxygen species/radical-scavenging activity. They also exhibited an enhanced tolerance of both salt and drought stress, which was associated with lower membrane permeability and a higher photosynthetic rate, respectively. Elevated carotenoid accumulation in RLE plants mitigated the reductions in leaf photosystem II efficiency and chlorophyll induced by abiotic stress. Expression of the carotenoid cleavage genes 9-cis-epoxycarotenoid dioxygenase, carotenoid cleavage dioxygenase 1 (CCD1) and CCD4 was higher in RLE plants, as was abscisic acid accumulation. IbLCY-ε silencing thus offers an effective approach for developing sweetpotato plants with increased tolerance to abiotic stress that will grow on global marginal lands with no reduction in nutritional value.

Functional Analysis of the Marigold (Tagetes erecta) Lycopene ε-cyclase (TeLCYe) Promoter in Transgenic Tobacco.

Lycopene ε-cyclases (LCYEs) are key enzymes in carotenoid biosynthesis converting red lycopene to downstream lutein. The flowers of marigold (Tagetes erecta) have been superior sources to supply lutein. However, the transcriptional regulatory mechanisms of LCYe in lutein synthesis are still unclear in marigold. In this work, the expression pattern of the TeLCYe gene in marigold indicated that TeLCYe mainly expressed in floral organs. To gain a better understanding of the expression and regulatory mechanism of TeLCYe gene, the TeLCYe promoter was isolated, sequenced, and analyzed through bioinformatics tools. The results suggested that the sequence of TeLCYe promoter contained various putative cis-acting elements responsive to exogenous and endogenous factors. The full-length TeLCYe promoter and three 5'-deletion fragments were fused to the GUS reporter gene and transferred into tobacco to test the promoter activities. A strong GUS activity was observed in stems of seedlings, leaves of seedlings, middle stems, top leaves, petals, stamens, and stigmas in transgenic tobacco containing full-length TeLCYe promoter LP0-2086. Deletion of - 910 to - 429 bp 5' to ATG significantly increased the GUS activity in chloroplast-rich tissues and floral organs, while deletion occurring from 1170 to 910 bp upstream ATG decreased the TeLCYe promoter strength in stems of seedlings, leaves of seedlings, top leaves and sepals. Functional characterization of the full-length TeLCYe promoter and its' deletion fragments in stable transgenic tobacco indicated that the LP0-2086 contains some specific cis-acting elements, which might result in the high-level expression of in floral organs, and LP2-910 might contain some specific cis-acting elements which improved GUS activities in vegetable tissues.

Overexpression of lycopene ε-cyclase gene from lycium chinense confers tolerance to chilling stress in Arabidopsis thaliana.

Lutein plays an important role in protecting the photosynthetic apparatus from photodamage and eliminating ROS to render normal physiological function of cells. As a rate-limiting step for lutein synthesis in plants, lycopene ε-cyclase catalyzes lycopene to δ-carotene. We cloned a lycopene ε-cyclase gene (Lcε-LYC) from Lycium chinense (L. chinense), a deciduous woody perennial halophyte growing in various environmental conditions. The Lcε-LYC gene has an ORF of 1569bp encoding a protein of 522 aa. The deduced amino acid sequence of Lcε-LYC gene has higher homology with LycEs in other plants, such as Nicotiana tabacum and Solanum tuberosum. When L. chinense was exposed to chilling stress, relative expression of Lcε-LYC increased. To study the protective role of Lcε-LYC against chilling stress, we overexpressed the Lcε-LYC gene in Arabidopsis thaliana. Lcε-LYC overexpression led to an increase of lutein accumulation in transgenic A. thaliana, and the content of lutein decreased when transgenics were under cold conditions. In addition, the transgenic plants under chilling stress displayed higher activities of superoxide dismutase (SOD) and peroxidase (POD) and less H2O2 and malondialdehyde (MDA) than the control. Moreover, the photosynthesis rate, photosystem II activity (Fv/fm), and Non-photochemical quenching (NPQ) also increased in the transgenetic plants. On the whole, overexpression of Lcε-LYC ameliorates photoinhibition and photooxidation, and decreases the sensitivity of photosynthesis to chilling stress in transgenic plants.

Molecular characterisation and the light-dark regulation of carotenoid biosynthesis in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.).

Seven partial-length cDNAs and 1 full-length cDNA that were involved in carotenoid biosynthesis and 2 partial-length cDNAs that encoded carotenoid cleavage dioxygenases were first isolated and characterised in 2 tartary buckwheat cultivars (Fagopyrum tataricum Gaertn.), Hokkai T8 and Hokkai T10. They were constitutively expressed at high levels in the leaves and flowers, where carotenoids are mostly distributed. During the seed development of tartary buckwheat, an inverse correlation between transcription level of carotenoid cleavage dioxygenase and carotenoid content was observed. The light-grown sprouts exhibited higher levels of expression of carotenoid biosynthetic genes in T10 and carotenoid content in both T8 and T10 compared to the dark-grown sprouts. The predominant carotenoids in tartary buckwheat were lutein and ß-carotene, and very abundant amounts of these carotenoids were found in light-grown sprouts. This study might broaden our understanding of the molecular mechanisms involved in carotenoid biosynthesis and indicates targets for increasing the production of carotenoids in tartary buckwheat.

Carotenoids, versatile components of oxygenic photosynthesis.

Carotenoids (CARs) are a group of pigments that perform several important physiological functions in all kingdoms of living organisms. CARs serve as protective agents, which are essential structural components of photosynthetic complexes and membranes, and they play an important role in the light harvesting mechanism of photosynthesizing plants and cyanobacteria. The protection against reactive oxygen species, realized by quenching of singlet oxygen and the excited states of photosensitizing molecules, as well as by the scavenging of free radicals, is one of the main biological functions of CARs. X-ray crystallographic localization of CARs revealed that they are present at functionally and structurally important sites of both the PSI and PSII reaction centers. Characterization of a CAR-less cyanobacterial mutant revealed that while the absence of CARs prevents the formation of PSII complexes, it does not abolish the assembly and function of PSI. CAR molecules assist in the formation of protein subunits of the photosynthetic complexes by gluing together their protein components. In addition to their aforementioned indispensable functions, CARs have a substantial role in the formation and maintenance of proper cellular architecture, and potentially also in the protection of the translational machinery under stress conditions.