Lycopene Cyclase Research Articles

Gene editing technology combined with response surface optimization to improve the synthesis ability of lycopene in Pantoea dispersa MSC14.

The aim of this study is to engineer Pantoea dispersa MSC14 into a strain capable of producing lycopene and to enhance its lycopene content. Our laboratory isolated the strain P. dispersa MSC14 from petroleum-contaminated soil in a mining area. Whole-genome sequencing confirmed the existence of a carotenoid synthesis pathway in this strain. This study employed an optimized CRISPR/Cas9 system to perform a traceless gene knockout of the lycopene cyclase gene crtY and to overexpress the octahydrolycopene dehydrogenase gene crtI in the P. dispersa MSC14. This strategic genetic modification successfully constructed the lycopene-producing strain MSC14-LY, which exhibited a notable lycopene content with a biomass productivity of 553μg of lycopene per gram dry cell weight(DCW). Additionally, the components of the lycopene fermentation medium were optimized using Plackett-Burman (PB) design and Response Surface Methodology (RSM). The average lycopene content was increased to 5.13mg g -1DCW in the optimized LY fermentation medium. Through genetic engineering, P. dispersa MSC14 was transformed into a strain capable of producing lycopene, achieving a yield of 5.13mg g-1 DCW after medium optimization. Genetic engineering successfully transformed P. dispersa MSC14 into a strain capable of producing lycopene, achieving a yield of 5.13mg g-1 DCW after medium optimization.

Genomic insights on carotenoid synthesis by extremely halophilic archaea Haloarcularubripromontorii BS2, Haloferaxlucentense BBK2 and Halogeometricumborinquense E3 isolated from the solar salterns of India

Haloarchaeal cultures were isolated from solar salterns of Goa and Tamil Nadu and designated as BS2, BBK2 and E3. These isolates grew with a characteristic bright orange to pink pigmentation and were capable of growing in media containing upto 25% (w/vol) NaCl. Whole genome sequencing (WGS) of the three haloarchaeal strains BS2, BBK2 and E3 indicated an assembled genomic size of 4.1 Mb, 3.8 Mb and 4 Mb with G + C content of 61.8, 65.6 and 59.8% respectively. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the archaeal isolates belong to Haloarcula, Haloferax and Halogeometricum genera. Haloarcula rubripromontorii BS2 was predicted to have 4292 genes with 4242 CDS regions, 46 tRNAs, 6 rRNAs and 3 misc_RNAs. In case of Haloferaxlucentense BBK2, 3840 genes with 3780 CDS regions were detected along with 52 tRNAs, 5 rRNAs and 3 misc_RNAs. Halogeometricumborinquense E3 contained 4101 genes, 4043 CDS regions, 52 tRNAs, 4 rRNAs, and 2 misc_RNAs. The functional annotation and curation of the haloarchaeal genome, revealed C50 carotenoid biosynthetic genes like phytoene desaturase/carotenoid 3′ -4′ desaturase (crtI), lycopene elongase (ubiA/lyeJ) and carotenoid biosynthesis membrane protein (cruF) in the three isolates. Whereas crtD (C-3′,4′ desaturase), crtY (lycopene cyclase) and brp/blh (β-carotene dioxygenase) genes were identified only in BS2.

Haematococcus lacustris Carotenogensis: A Historical Event of Primary to Secondary Adaptations to Earth's Oxygenation.

(1) Background: Oxygen has exerted a great effect in shaping the environment and driving biological diversity in Earth's history. Green lineage has evolved primary and secondary carotenoid biosynthetic systems to adapt to Earth's oxygenation, e.g., Haematococcus lacustris, which accumulates the highest amount of secondary astaxanthin under stresses. The two systems are controlled by lycopene ε-cyclase (LCYE) and β-cyclase (LCYB), which leave an important trace in Earth's oxygenation. (2) Objectives: This work intends to disclose the underlying molecular evolutionary mechanism of Earth's oxygenation in shaping green algal carotenogensis with a special focus on lycopene cyclases. (3) Methods: The two kinds of cyclases were analyzed by site-directed mutagenesis, phylogeny, divergence time and functional divergence. (4) Results: Green lineage LCYEs appeared at ~1.5 Ga after the first significant appearance and accumulation of atmospheric oxygen, the so-called Great Oxygenation Event (GOE), from which LCYBs diverged by gene duplication. Bacterial β-bicyclases evolved from β-monocyclase. Enhanced catalytic activity accompanied evolutionary transformation from ε-/β-monocyclase to β-bicyclase. Strong positive selection occurred in green lineage LCYEs after the GOE and in algal LCYBs during the second oxidation, the Neoproterozoic Oxygenation Event (NOE). Positively selected sites in the catalytic cavities of the enzymes controlled the mono-/bicyclase activity, respectively. Carotenoid profiling revealed that oxidative adaptation has been wildly preserved in evolution. (5) Conclusions: the functionalization of the two enzymes is a result of primary to secondary adaptations to Earth's oxygenation.

Gene structure and potential regulation of the lycopene cyclase genes in Bixa orellana L.

The online version contains supplementary material available at 10.1007/s12298-023-01384-8.

TgLCYB1 regulated by TgWRKY22 enhances the tolerance of Torreya grandis to waterlogging stress

β-Carotene functions in plant growth and development and plays an important role in resisting abiotic stress, such as drought and salt stress. The specific function and mechanism by which β-carotene responds to waterlogging stress, however, remain elusive. In this study, we found that β-carotene content and lycopene cyclase (TgLCYB1) expression, both in leaves and roots of Torreya grandis, were increased under waterlogging treatment. Subcellular localization assays indicated that TgLCYB1 was localized in the chloroplasts. Phenotypic, physiological, and metabolome analysis showed that overexpression of TgLCYB1 enhanced the tolerance of tomato plants to waterlogging stress. Furthermore, application of a LCYB enzyme inhibitor, 2-(4-chlorophenylthio)-triethylamine hydrochloride, markedly enhanced the sensitivity of T. grandis to waterlogging stress. In addition, yeast one-hybrid assay, the dual luciferase assay system, and real-time quantitative PCR indicated that waterlogging stress induced TgWRKY22 to increase TgLCYB1 expression by binding to the TgLCYB1 promoter. Collectively, our results indicated that TgWRKY22 positively regulated TgLCYB1 expression to improve the activities of antioxidant enzyme and increase the levels of some key metabolites, thereby relieving waterlogging-induced oxidative damage, and consequently modulating the waterlogging stress response. This study contributes to a more comprehensive understanding of carotenoid functions and the role LCYB genes play in plant stress response.

Molecular mechanism of lycopene cyclases regulating carotenoids ratio in different branches during tea leaf and flower development

Carotenoids are essential components in tea quality, contributing to leaf color and aroma. However, little information about carotenoids in different tea cultivars and their biosynthesis regulation mechanism during leaf development is known. Here we analyzed carotenoids by HPLC in the buds and leaves of 113 tea cultivars harvested on the same day. By profile clustering, carotenoids were divided into five groups. Same group cultivars displayed divergence in the total content of carotenoids but a similar molar ratio. To figure out the molecular mechanisms of this phenomenon, we further characterized all functional lycopene cyclases, which are the branch point of the carotenoid biosynthesis pathway. Two β-lycopene cyclases (CsLCYB1 and CsLCYB2) and one ε-lycopene cyclase (CsLCYE1) were cloned. Subcellular localization analysis showed that all cloned CsLCYs were localized in plastids. Enzyme activity assays in E. coli indicated both CsLCYBs catalyzed lycopene into β-carotene, and CsLCYE1 produced δ-carotene and ε-carotene. We found CsLCYB1 and CsLCYE1 predominantly expressed in leaf, while CsLCYB2 was mainly expressed during flowering stages. Suppression by antisense oligonucleotides reduced CsLCYB1 and CsLCYE1 transcripts and led to reduction of both β,β-branch and β,ε-branch carotenoids in leaf. The expression levels of CsLCYB1 showed a significant positive correlation with β,β-branch carotenoids in leaf. Our study provides carotenoid profiles of different tea cultivars, which can assist tea producers in selecting cultivars of interest. Meanwhile, we proposed the molecular mechanism of carotenoids reflecting the tenderness of tea plant leaf from a metabolic flux perspective, and suggested lycopene cyclase that could be applied to the breeding of tea varieties with different branch carotenoids.

Enhancement of β-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene β-cyclase

β-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of β-carotene and α-carotene. Aiming to develop the microalgae with enhanced β-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of β-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant β-carotene), confirming the superior activity of CrtY on β-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased β-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

Functional Characterization of Lycopene β- and ε-Cyclases from a Lutein-Enriched Green Microalga Chlorella sorokiniana FZU60.

Lutein is a high-value carotenoid with many human health benefits. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently β-cyclize both ends of lycopene to produce β-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into β-monocyclic γ-carotene and ultimately produced α-carotene with a β-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.

Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis

Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and β-carotene hydroxylase (crtZ) from Pantoea ananatis to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer Escherichia coli strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites.This figure was drawn using BioRender ( https://www.biorender.com ).

Zeaxanthin is required for eyespot formation and phototaxis in Euglena gracilis.

The eyespot apparatus is an organelle that forms carotenoid-rich globules in diverse flagellated microalgae and functions in phototaxis. The euglenophytes have structurally and functionally distinct eyespot apparatuses from chlorophytes. β-Carotene is the most abundant pigment detected in chlorophytes' eyespots, while xanthophylls such as zeaxanthin and diadinoxanthin have been suggested to function in euglenophytes' eyespots. Here, we investigated the association between carotenoid composition and eyespot formation via pathway-scale mutagenesis using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing in the euglenophyte Euglena gracilis. Lycopene cyclase (lcy) mutants exhibited sole lycopene accumulation, defective red eyespots, and phototactic insensitivity. Conversely, β-carotene hydroxylase (cytochrome P450 97h1, cyp97h1) mutants accumulated β-carotene and its hydroxylated products β-cryptoxanthin and zeaxanthin and formed phototactic eyespot apparatuses, while cyp97h1 cyp97f2 double mutants were deficient in β-carotene hydroxylation and mostly lacked functional eyespots. Thus, zeaxanthin is required for the stable formation of functional eyespots in E. gracilis, highlighting evolutionary differences between euglenophytes and chlorophytes in the metabolic regulation of photoreactive organelle formation.

Hierarchical dynamic regulation of Saccharomyces cerevisiaefor 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.

Concurrent Production of α- and β-Carotenes with Different Stoichiometries Displaying Diverse Antioxidative Activities via Lycopene Cyclases-Based Rational System.

α- and β-carotenes belong to the most essential carotenoids in the human body and display remarkable pharmacological value for health due to their beneficial antioxidant activities. Distinct high α-/β-carotene stoichiometries have gained increasing attention for their effective preventions of Alzheimer's disease, cardiovascular disease, and cancer. However, it is extremely difficult to obtain α-carotene in nature, impeding the accumulations of high α-/β-carotene stoichiometries and excavation of their antioxidant activities. Herein, we developed a dynamically operable strategy based on lycopene cyclases (LCYB and LCYE) for concurrently enriching α- and β-carotenes along with high stoichiometries in E. coli. Membrane-targeted and promoter-centered approaches were firstly implemented to spatially enhance catalytic efficiency and temporally boost expression of TeLCYE to address its low competitivity at the starting stage. Dynamically temperature-dependent regulation of TeLCYE and TeLCYB was then performed to finally achieve α-/β-carotene stoichiometries of 4.71 at 37 °C, 1.65 at 30 °C, and 1.06 at 25 °C, respectively. In the meantime, these α-/β-carotene ratios were confirmed to result in diverse antioxidative activities. According to our knowledge, this is the first time that both the widest range and antioxidant activities of high α/β-carotene stoichiometries were reported in any organism. Our work provides attractive potentials for obtaining natural products with competitivity and a new insight on the protective potentials of α-/β-carotenes with high ratios for health supply.

Carotenoid analysis and functional characterization of lycopene cyclases in Zinnia elegans L.

Carotenoids, a group of essential pigments for petal coloration in many species, could be generally categorized into linear and cyclic groups. The cyclization of the linear substrate lycopene at both end groups by β- and ε-cyclases (LCYBs and LCYEs, respectively) is a key branching point of the biosynthesis of cyclic carotenoids in flowers. Common zinnia (Zinnia elegans L.), an Asteraceae family plant that has been widely used in landscape greening, is best known for its brilliant flower colors. However, the mechanism of its petal coloration is not well studied, especially with respect to carotenoid pigmentation. In this study, total carotenoid content and carotenoid compositions of petals were analyzed in two common zinnia cultivars, 'Dreamland Red’ (DRE) and 'Dreamland Yellow’ (DY). Despite the lighter color of DY petals, the total carotenoid content in DY flowers was significantly higher than that in DRE. Metabolomic analysis revealed that the major carotenoids of both cultivars are cyclic carotenoids, with β-carotene being the most abundant. In addition, one LCYB, LCYE, and capsanthin/capsorubin synthase (CCS) gene were identified from Z. elegans by homology-based search in transcriptome. After cloning and sequencing, full-length open reading frame of each gene is exactly the same between two cultivars. Bacterial pigment complementation experiments revealed that ZeLCYB and ZeCCS can cyclize lycopene at both ends to produce β-carotene. Unlike most LCYEs characterized from higher plants, ZeLCYE produced predominantly ε-carotene in E. coli system. However, ZeLCYE is likely to have a lower affinity for lycopene or lower activity compared with ZeLCYB. Gene expression analysis showed that DY has higher expression levels of ZeLCYB and ZeCCS than DRE at specific developmental stages, consistent with its higher total carotenoid content. This work would lay a solid foundation for future studies on carotenoid accumulation in Z. elegans.

DsLCYB Directionally Modulated β-Carotene of the Green Alga Dunaliella salina under Red Light Stress.

Carotenoids, which are natural pigments found abundantly in wide-ranging species, have diverse functions and high industrial potential. The carotenoid biosynthesis pathway is very complex and has multiple branches, while the accumulation of certain metabolites often affects other metabolites in this pathway. The DsLCYB gene that encodes lycopene cyclase was selected in this study to evaluate β-carotene production and the accumulation of β-carotene in the alga Dunaliella salina. Compared with the wild type, the transgenic algal species overexpressed the DsLCYB gene, resulting in a significant enhancement of the total carotenoid content, with the total amount reaching 8.46 mg/g for an increase of up to 1.26-fold. Interestingly, the production of α-carotene in the transformant was not significantly reduced. This result indicated that the regulation of DsLCYB on the metabolic flux distribution of carotenoid biosynthesis is directional. Moreover, the effects of different light-quality conditions on β-carotene production in D. salina strains were investigated. The results showed that the carotenoid components of β-carotene and β-cryptoxanthin were 1.8-fold and 1.23-fold higher than that in the wild type under red light stress, respectively. This suggests that the accumulation of β-carotene under red light conditions is potentially more profitable.

A comprehensive analysis of carotenoids metabolism in two red-fleshed mutants of Navel and Valencia sweet oranges (Citrus sinensis).

Kirkwood Navel and Ruby Valencia are two spontaneous bud mutations of the respective parental lines of sweet orange (Citrus sinensis) Palmer Navel and Olinda Valencia, showing an atypical red pigmentation of the pulp. These red-fleshed varieties are commercially available and highly attractive for consumers but their carotenoid metabolism and the basis of the mutation have not been investigated. The red colour of Kirkwood and Ruby pulp was observed from the very early stages of fruit development until full maturity and associated with an altered carotenoid profiling. The red-fleshed varieties accumulated from 6- up to 1000-times more total carotenoids compared to the standard oranges. Specifically, the pulp of Kirkwood and Ruby accumulated large amounts of phytoene and phytofluene, and moderate contents of lycopene. Moreover, the red-fleshed oranges contained other unusual carotenes as δ-carotene, and lower concentrations of downstream products such as β,β-xanthophylls, abscisic acid (ABA) and ABA-glucosyl ester. This peculiar profile was associated with chromoplasts with lycopene crystalloid structures and round vesicles likely containing colourless carotenes. The flavedo and leaves of Kirkwood and Ruby showed minor changes in carotenoids, mainly limited to higher levels of phytoene. The carotenoid composition in Kirkwood and Ruby fruits was not explained by differences in the transcriptional profile of 26 genes related to carotenoid metabolism, covering the main steps of biosynthesis, catabolism and other processes related to carotenoid accumulation. Moreover, sequence analysis of the lycopene cyclase genes revealed no alterations in those of the red-fleshed oranges compared to the genes of the standard varieties. A striking event observed in Kirkwood and Ruby trees was the reddish coloration of the inner side of the bark tissue, with larger amounts of phytoene, accumulation of lycopene and lower ABA content. These observation lead to the conclusion that the mutation is not only manifested in fruit, affecting other carotenogenic tissues of the mutant plants, but with different consequences in the carotenoid profile. Overall, the carotenoid composition in the red-fleshed mutants suggests a partial blockage of the lycopene β-cyclization in the carotenoid pathway, rendering a high accumulation of carotenes upstream lycopene and a reduced flow to downstream xanthophylls and ABA.

Hybrid histidine kinase HisK2301 modulates carotenoid production to counteract cold-induced oxidative stress in Rhodosporidium kratochvilovae YM25235 under low temperature.

Hybrid histidine kinases (HHKs) are major sensor proteins for fungi that contribute to stress tolerance. In the present work, we investigated the roles and mechanisms of the HHK HisK2301 in cold-adapted Rhodosporidium kratochvilovae strain YM25235. The HisK2301 deletion strain was constructed by homologous recombination method and arranged for multiple stress tests. We analysed the content of carotenoid using UV-Vis and HPLC. Relative transcript levels of genes phytoene desaturase (RKCrtI) and phytoene synthase and lycopene cyclase (RKCrtYB) were analysed by RT-qPCR. Intracellular reactive oxygen species (ROS) generation was measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Our results clearly indicated that YM25235 produces γ-carotene, torulene, β-carotene and torularhodin, with the latter two components strongly related to adapt to cold. HisK2301 is crucial for YM25235 adaptation to different types of stress such as cold, salt, osmotic and oxidative stress. Growth at low temperature clearly induced oxidative stress in YM25235, as more ROS accumulated at cold. During cold stress, HisK2301 is suggested to sense cold-induced ROS signals and then promote carotenoid production partially by RKCrtI and RKCrtYB to scavenge excessive ROS production. Such an inducible protective system may confer YM25235 fast response and better adaptation to cold stress. To conclude, our findings give the first insight into the effect of HisK2301 on carotenoid biosynthesis and cold-induced oxidative stress in fungi under low temperature and suggest the potential use of the cold-adapted HHK HisK2301 in industrial production of carotenoid.

Crucial carotenogenic genes elevate hyperaccumulation of both fucoxanthin and β-carotene in Phaeodactylum tricornutum

Burgeoning demand for carotenoids from natural resources has urged to explore the promising metabolic engineering strategy to improve algal systems. However, existing biological intricacies in algal carotenogenesis warrant identification and exploitation of potential molecular candidate(s) for metabolic engineering to exploit the maximum potential of microalgal cell factories. Here, we identified two critical carotenogenic genes, including 1-deoxy-d-xylulose 5-phosphate synthase and lycopene cyclase and concurrently overexpressed in Phaeodactylum tricornutum. Molecular analysis proved the successful integration and expression of these two genes in engineered algal cells. Dual overexpression concurrently elevated fucoxanthin and β-carotene content up to 6.53 mg/g and 4.34 mg/g dry cell weight, respectively, which is the highest reported content of two carotenoids simultaneously, particularly the highest β-carotene content. Besides, dual overexpression did not impede algal physiological properties and biomass production. The findings exemplified the potential metabolic candidates capable of elevating the complex carotenogenesis and demonstrated a promising strategy to overcome the bottleneck in the commercial production of algal carotenoids.

Functional Characterization of Two Lycopene Cyclases from Sweet Osmanthus (Osmanthus fragrans)

• LCYB from sweet osmanthus can efficiently β -cyclize lycopene to produce β-carotene. • LCYE from sweet osmanthus is able to cyclize lycopene to produce bicyclic ε-carotene. Sweet osmanthus ( Osmanthus fragrans ) flowers, as a food flavor additive, are rich in carotenoids, the majority of which are cyclized carotenoids. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of the open ends of acyclic lycopene and manipulate the metabolic flux into different downstream branches. Although both LCYB and LCYE have been fully characterized in many plants, little information is available concerning the functions of sweet osmanthus LCYB and LCYE in carotenoid biosynthesis. In this study, we cloned two lycopene cyclase genes OfLCYB and OfLCYE in sweet osmanthus. The expression levels of OfLCYB in the petals gradually increased during flower opening, having a significant positive correlation with total carotenoid contents in each sweet osmanthus cultivar. Pigment complementation assay in Escherichia coli demonstrated that OfLCYB could efficiently β-cyclize both ends of lycopene to produce β-carotene (β,β-carotene), while OfLCYE was able to catalyze the formation of ε-carotene (ε,ε-carotene) at both ends of lycopene. The characterization of LCYB and LCYE from sweet osmanthus drives the first bifurcation step toward β-carotene and α-carotene. Our results will lay theoretical foundation for future metabolic engineering of carotenoid biosynthesis and accumulation in sweet osmanthus flowers.

Removal of lycopene substrate inhibition enables high carotenoid productivity in Yarrowia lipolytica

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica. To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive β-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L β-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products.