Metabolic Engineering of Candida tropicalis for the De Novo Synthesis of ß-Ionone.

ß-ionone, a norisoprenoid, is a natural aromatic compound derived from plants, which displays various biological activities including anticancer, antioxidant and deworming properties. Due to its large biomass and strong environmental tolerance, the nonconventional oleaginous yeast Candida tropicalis was selected to efficiently synthesize ß-ionone. We initially investigated the capacity of the cytoplasm and subcellular compartments to synthesize ß-ionone independently. Subsequently, through adaptive screening of enzymes, functional identification of subcellular localization signal peptides and subcellular compartment combination strategies, a titer of 152.4 mg/L of ß-ionone was achieved. Finally, directed evolution of rate-limiting enzyme and overexpression of key enzymes were performed to enhance ß-ionone production. The resulting titer was 400.5 mg/L in shake flasks and 730 mg/L in a bioreactor. This study demonstrates the first de novo synthesis of ß-ionone in C. tropicalis, providing a novel cellular chassis for terpenoid fragrances with considerable industrial potential.

Temporal Dynamics and Influential Factors of Taste and Odor Compounds in the Eastern Drinking Water Source of Chaohu Lake, China: A Comparative Analysis of Global Freshwaters.

The escalating proliferation of cyanobacteria poses significant taste and odor (T/O) challenges, impacting freshwater ecosystems, public health, and water treatment costs. We examined monthly variations in four T/O compounds from September 2011 to August 2012 in Chaohu Lake's eastern drinking water source (DECL). More importantly, we compared the reported T/O occurrence and the related factors in freshwater bodies worldwide. The assessment of T/O issues indicated a severe and widespread problem, with many cases surpassing odor threshold values. Remarkably, China reported the highest frequency and severity of odor-related problems. A temporal analysis revealed variations in odor occurrences within the same water body across different years, emphasizing the need to consider high values in all seasons for water safety. Globally, T/O issues were widespread, demanding attention to variations within the same water body and across different layers. Algae were crucial contributors to odor compounds, necessitating targeted interventions due to diverse odorant sources and properties. A correlation analysis alone lacked definitive answers, emphasizing the essential role of further validation, such as algae isolation. Nutrients are likely to have influenced the T/O, as GSM and MIB correlated positively with nitrate and ammonia nitrogen in DECL, resulting in proposed control recommendations. This study offers recommendations for freshwater ecosystem management and serves as a foundation for future research and management strategies to address T/O challenges.

Characterization of YABBY transcription factors in Osmanthus fragrans and functional analysis of OfYABBY12 in floral scent formation and leaf morphology.

BACKGROUND: The plant-specific YABBY transcription factor family plays important roles in plant growth and development, particularly leaf growth, floral organ formation, and secondary metabolite synthesis. RESULTS: Here, we identified a total of 13 OfYABBY genes from the Osmanthus fragrans genome. These 13 OfYABBY genes were divided into five subfamilies through phylogenetic analysis, and genes in the same subfamily showed similar gene structures and conserved protein motifs. Gene duplication promoted the expansion of the OfYABBY family in O. fragrans. Tissue-specific expression analysis showed that the OfYABBY family was mainly expressed in O. fragrans leaves and floral organs. To better understand the role of OfYABBY genes in plant growth and development, OfYABBY12 was selected for heterologous stable overexpression in tobacco, and OfYABBY12-overexpressing tobacco leaves released significantly fewer volatile organic compounds than wild-type tobacco leaves. Overexpression of OfYABBY12 led to the downregulation of NtCCD1/4 and decreased ß-ionone biosynthesis. Correspondingly, a dual-luciferase assay showed that OfYABBY12 negatively regulated the expression of OfCCD4, which promotes ß-ionone synthesis. Furthermore, tobacco leaves overexpressing OfYABBY12 were curled and wrinkled and had significantly reduced leaf thickness and leaf inclusions and significantly extended flower pistils (styles). CONCLUSION: Overall, the results suggest that the OfYABBY gene family may influence the biosynthesis of the floral scent (especially ß-ionone) in O. fragrans and may regulate leaf morphogenesis and lateral organs.

The effect of ß-ionone on bacterial cells: the use of specific lux-biosensors.

The diversity of the biological activity of volatile organic compounds (VOCs), including unsaturated ketone ß-ionone, promising pharmacological, biotechnological, and agricultural agent, has aroused considerable interest. However, the functional role and mechanisms of action of VOCs remain insufficiently studied. In this work, the response of bacterial cells to the action of ß-ionone was studied using specific bioluminescent lux-biosensors containing stress-sensitive promoters. We determined that in Escherichia coli cells, ß-ionone induces oxidative stress (PkatG and Pdps promoters) through a specific response mediated by the OxyR/OxyS regulon, but not SoxR/SoxS (PsoxS promoter). It has been shown that ß-ionone at high concentrations (50 µM and above) causes a weak induction of the expression from the PibpA promoter and slightly induces the PcolD promoter in the E. coli biosensors; the observed effect is enhanced in the ΔoxyR mutants. This indicates the presence of some damage to proteins and DNA. ß-Ionone was found to inhibit the bichaperone-dependent DnaKJE-ClpB refolding of heat-inactivated bacterial luciferase in E. coli wild-type and ΔibpB mutant strains. In the cells of the Gram-positive bacterium Bacillus subtilis 168 pNK-MrgA ß-ionone does not cause oxidative stress. Thus, in this work, the specificity of bacterial cell stress responses to the action of ß-ionone was shown.

Engineering a non-model yeast Rhodotorula mucilaginosa for terpenoids synthesis.

Terpenoids have tremendous biological activities and are widely employed in food, healthcare and pharmaceutical industries. Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction. The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids, indicating a strong endogenous isoprene pathway with readily available metabolic intermediates. This study constructed several engineered strains of R. mucilaginosa with the aim of producing different terpenoids. Monoterpene α-terpineol was produced by expressing the α-terpineol synthase from Vitis vinifera. The titer of α-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway. Overexpression of α-farnesene synthase from Malus domestica, in combination with MVA pathway rate-limiting gene resulted in significant increase in α-farnesene production, reaching a titer of 822 mg/L. The carotenoid degradation product ß-ionone was produced at a titer of 0.87 mg/L by expressing the ß-ionone synthase from Petunia hybrida. This study demonstrates the potential of R. mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.

Integration of rice apocarotenoid profile and expression pattern of Carotenoid Cleavage Dioxygenases reveals a positive effect of ß-ionone on mycorrhization.

Carotenoids are susceptible to degrading processes initiated by oxidative cleavage reactions mediated by Carotenoid Cleavage Dioxygenases that break their backbone, leading to products called apocarotenoids. These carotenoid-derived metabolites include the phytohormones abscisic acid and strigolactones, and different signaling molecules and growth regulators, which are utilized by plants to coordinate many aspects of their life. Several apocarotenoids have been recruited for the communication between plants and arbuscular mycorrhizal (AM) fungi and as regulators of the establishment of AM symbiosis. However, our knowledge on their biosynthetic pathways and the regulation of their pattern during AM symbiosis is still limited. In this study, we generated a qualitative and quantitative profile of apocarotenoids in roots and shoots of rice plants exposed to high/low phosphate concentrations, and upon AM symbiosis in a time course experiment covering different stages of growth and AM development. To get deeper insights in the biology of apocarotenoids during this plant-fungal symbiosis, we complemented the metabolic profiles by determining the expression pattern of CCD genes, taking advantage of chemometric tools. This analysis revealed the specific profiles of CCD genes and apocarotenoids across different stages of AM symbiosis and phosphate supply conditions, identifying novel reliable markers at both local and systemic levels and indicating a promoting role of ß-ionone in AM symbiosis establishment.

The apocarotenoid ß-ionone regulates the transcriptome of Arabidopsis thaliana and increases its resistance against Botrytis cinerea.

Carotenoids are isoprenoid pigments indispensable for photosynthesis. Moreover, they are the precursor of apocarotenoids, which include the phytohormones abscisic acid (ABA) and strigolactones (SLs) as well as retrograde signaling molecules and growth regulators, such as ß-cyclocitral and zaxinone. Here, we show that the application of the volatile apocarotenoid ß-ionone (ß-I) to Arabidopsis plants at micromolar concentrations caused a global reprogramming of gene expression, affecting thousands of transcripts involved in stress tolerance, growth, hormone metabolism, pathogen defense, and photosynthesis. This transcriptional reprogramming changes, along with induced changes in the level of the phytohormones ABA, jasmonic acid, and salicylic acid, led to enhanced Arabidopsis resistance to the widespread necrotrophic fungus Botrytis cinerea (B.c.) that causes the gray mold disease in many crop species and spoilage of harvested fruits. Pre-treatment of tobacco and tomato plants with ß-I followed by inoculation with B.c. confirmed the effect of ß-I in increasing the resistance to this pathogen in crop plants. Moreover, we observed reduced susceptibility to B.c. in fruits of transgenic tomato plants overexpressing LYCOPENE ß-CYCLASE, which contains elevated levels of endogenous ß-I, providing a further evidence for its effect on B.c. infestation. Our work unraveled ß-I as a further carotenoid-derived regulatory metabolite and indicates the possibility of establishing this natural volatile as an environmentally friendly bio-fungicide to control B.c.

Organ-specific volatiles from Sonoran desert Krameria flowers as potential signals for oil-collecting bees.

The evolution of flowers that offer oils as rewards and are pollinated by specialized bees represents a distinctive theme in plant-pollinator co-diversification. Some plants that offer acetylated glycerols as floral oils emit diacetin, a volatile by-product of oil metabolism, which is utilized by oil-collecting bees as an index signal for the presence of floral oil. However, floral oils in the genus Krameria (Krameriaceae) contain ß-acetoxy-substituted fatty acids instead of acetylated glycerols, making them unlikely to emit diacetin as an oil-bee attractant. We analyzed floral headspace composition from K. bicolor and K. erecta, native to the Sonoran Desert of southwestern North America, in search of alternative candidates for volatile index signals. Using solid-phase microextraction, combined with gas chromatography-mass spectrometry, we identified 26 and 45 floral volatiles, respectively, from whole flowers and dissected flower parts of these two Krameria species. As expected, diacetin was not detected. Instead, ß-ionone emerged as a strong candidate for an index signal, as it was uniquely present in dissected oil-producing floral tissues (elaiophores) of K. bicolor, as well as the larval cells and provisions from its oil-bee pollinator, Centris cockerelli. This finding suggests that the floral oil of K. bicolor is perfused with ß-ionone in its tissue of origin and retains the distinctive raspberry-like scent of this volatile after being harvested by C. cockerelli bees. In contrast, the elaiophores of K. erecta, which are not thought to be pollinated by C. cockerelli, produced a blend of anise-related oxygenated aromatics not found in the elaiophores of K. bicolor. Our findings suggest that ß-ionone has the potential to impact oil-foraging by C. cockerelli bees through several potential mechanisms, including larval imprinting on scented provisions or innate or learned preferences by foraging adults.

Cyanobacterial VOCs ß-ionone and ß-cyclocitral poisoning Lemna turionifera by triggering programmed cell death.

ß-Ionone and ß-cyclocitral are two typical components in cyanobacterial volatiles, which can poison aquatic plants and even cause death. To reveal the toxic mechanisms of the two compounds on aquatic plants through programmed cell death (PCD), the photosynthetic capacities, caspase-3-like activity, DNA fragmentation and ladders, as well as expression of the genes associated with PCD in Lemna turionifera were investigated in exposure to ß-ionone (0.2 mM) and ß-cyclocitral (0.4 mM) at lethal concentration. With prolonging the treatment time, L. turionifera fronds gradually died, and photosynthetic capacities gradually reduced and even disappeared at the 96th h. This demonstrated that the death process might be a PCD rather than a necrosis, due to the gradual loss of physiological activities. When L. turionifera underwent the death, caspase-3-like was activated after 3 h, and reached to the strongest activity at the 24th h. TUNEL-positive nuclei were detected after 12 h, and appeared in large numbers at the 48th h. The DNA was cleaved by Ca2+-dependent endonucleases and showed obviously ladders. In addition, the expression of 5 genes (TSPO, ERN1, CTSB, CYC, and ATR) positively related with PCD initiation was up-regulated, while the expression of 2 genes (RRM2 and TUBA) negatively related with PCD initiation was down-regulated. Therefore, ß-ionone and ß-cyclocitral can poison L. turionifera by adjusting related gene expression to trigger PCD.

Antiplasmodial Activity of ß-Ionone and the Effect of the Compound on Amelioration of Anaemia and Oxidative Organ Damage in Mice Infected with Plasmodium berghei.

INTRODUCTION: Owing to evolution of parasite strains that are resistant to existing antimalarial drugs, research for novel antimalarial medicines is progressing on numerous fronts. PURPOSE: Herein, we evaluated the in vivo anti-Plasmodium berghei activity of ß-ionone including its ameliorative potential towards P. berghei-associated anaemia and oxidative organ damage. METHODS: Mice were infected with chloroquine-sensitive strain of P. berghei and then treated with ß-ionone at doses of 10 and 20 mg/kg body weight (BW) for seven days. The parasitemia, packed cell volume and redox sensitive biomarkers in the liver, brain and spleen were estimated. RESULTS: Our result showed that ß-ionone, in a dose-dependent fashion, significantly (p < 0.05) repressed the multiplication of P. berghei. More so, the compound, at doses of 10 and 20 mg/kg BW, significantly (p < 0.05) mitigated anaemia and organ damage induced by P. berghei. CONCLUSION: Overall, the findings demonstrated that ß-ionone has antiplasmodial actions and plays a mitigative role against P. berghei-induced anaemia and oxidative organ damage.

Preparation of ß-ionone microcapsules by gelatin/pectin complex coacervation.

ß-ionone has a unique violet odor and good biological activity, which is an essential fragrance component and potential anticancer drug. In this paper, ß-ionone was encapsulated using complex coacervation of gelatin and pectin, followed by cross-linking with glutaraldehyde. The pH value, wall material concentration, core-wall ratio, homogenization conditions, and curing agent content were investigated in the single-factor experiments. For example, the encapsulation efficiency increased with the homogenization speed, which reached a relatively high value at 13000 r/min for 5 min. The gelatin/pectin ratio (3:1, w/w) and pH value (4.23) significantly affected the size, shape, and encapsulation efficiency of the microcapsule. The fluorescence microscope and SEM were used to characterize the morphology of the microcapsules, in which the microcapsule has a stable morphology, uniform size, and spherical multinuclear structure. FTIR measurements confirmed the electrostatic interactions between gelatin and pectin during complex coacervation. Thermogravimetric analysis (TGA) revealed that the microcapsules could maintain good thermal stability over 260 °C. The release rate of ß-ionone microcapsule was only 20.6 % after 30 days at the low temperature of 4 °C. These findings provide an effective carrier to deliver flavors like ß-ionone and could be useful in the fields of daily chemicals and textiles.

Co-Occurrence of Taste and Odor Compounds and Cyanotoxins in Cyanobacterial Blooms: Emerging Risks to Human Health?

Cyanobacteria commonly form large blooms in waterbodies; they can produce cyanotoxins, with toxic effects on humans and animals, and volatile compounds, causing bad tastes and odors (T&O) at naturally occurring low concentrations. Notwithstanding the large amount of literature on either cyanotoxins or T&O, no review has focused on them at the same time. The present review critically evaluates the recent literature on cyanotoxins and T&O compounds (geosmin, 2-methylisoborneol, ß-ionone and ß-cyclocitral) to identify research gaps on harmful exposure of humans and animals to both metabolite classes. T&O and cyanotoxins production can be due to the same or common to different cyanobacterial species/strains, with the additional possibility of T&O production by non-cyanobacterial species. The few environmental studies on the co-occurrence of these two groups of metabolites are not sufficient to understand if and how they can co-vary, or influence each other, perhaps stimulating cyanotoxin production. Therefore, T&Os cannot reliably serve as early warning surrogates for cyanotoxins. The scarce data on T&O toxicity seem to indicate a low health risk (but the inhalation of ß-cyclocitral deserves more study). However, no data are available on the effects of combined exposure to mixtures of cyanotoxins and T&O compounds and to combinations of T&O compounds; therefore, whether the co-occurrence of cyanotoxins and T&O compounds is a health issue remains an open question.

Purification and biochemical characterization of a novel ene- reductase from Kazachstania exigua HSC6 for dihydro-ß-ionone from ß-ionone.

PURPOSE: We purified and characterized a novel ene-reductase (KaDBR1) from Kazachstania exigua HSC6 for the synthesis of dihydro-ß-ionone from ß-ionone. METHODS: KaDBR1 was purified to homogeneity by ammonium sulfate precipitation and phenyl-Sepharose Fast Flow and Q-Sepharose chromatography. The purified enzyme was characterized by measuring the amount of dihydro-ß-ionone from ß-ionone with LC-MS analysis method. RESULTS: The molecular mass of KaDBR1 was estimated to be 45 kDa by SDS-PAGE. The purified KaDBR1 enzyme had optimal activity at 60 °C and pH 6.0. The addition of 5 mM Mg2+, Ca2+, Al3+, Na+, and dithiothreitol increased the activity of KaDBR1 by 25%, 18%, 34%, 20%, and 23%, respectively. KaDBR1 favored NADH over NADPH as a cofactor, and its catalytic efficiency (kcat/Km) toward ß-ionone using NADH was 8.1-fold greater than when using NADPH. CONCLUSION: Owing to its unique properties, KaDBR1 is a potential candidate for the enzymatic biotransformation of ß-ionone to dihydro-ß-ionone in biotechnology applications.

Analysis of apocarotenoid volatiles from lettuce (Lactuca sativa) induced by insect herbivores and characterization of carotenoid cleavage dioxygenase gene.

Plant apocarotenoids have been shown to have a diverse biological role in herbivore-plant interactions. Despite their importance, little is known about herbivores' effect on apocarotenoid emissions in Lactuca sativa. In this study, we examined changes in apocarotenoid emissions in lettuce leaves after infestation by two insects, viz., Spodoptera littoralis larvae and Myzus persicae aphids. We found that ß-ionone and ß-cyclocitral showed higher concentrations than the other apocarotenoids, with a significant increase as per the intensity of infestation of both herbivore species. Furthermore, we performed functional characterization of Lactuca sativa carotenoid cleavage dioxygenase 1 (LsCCD1) genes. Three LsCCD1 genes were overexpressed in E. coli strains, and recombinant proteins were assayed for cleavage activity on an array of carotenoid substrates. The LsCCD1 protein cleaved ß-carotene at the 9,10 (9',10') positions producing ß-ionone. The transcript analysis of LsCCD1 genes revealed differential expression patterns under varying levels of herbivores' infestation, but the results were inconsistent with the pattern of ß-ionone concentrations. Our results suggest that LsCCD1 is involved in the production of ß-ionone, but other regulatory factors might be involved in its induction in response to herbivory. These results provide new insights into apocarotenoid production in response to insect herbivory in lettuce. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03511-4.

ß-Ionone represses renal cell carcinoma progression through activating LKB1/AMPK-triggered autophagy.

ß-Ionone, the end ring analog of ß-carotenoids, has been proven to have an antitumor effect in a variety of cancers. In this study, we investigated the impact of ß-ionone on renal cell carcinoma (RCC) cell lines (786-O and ACHN) using colony formation assays, flow cytometry analysis, and western blot analysis. We found that ß-ionone effectively inhibited the proliferation of RCC cells in vitro, which was also confirmed in a xenograft model. Moreover, we found that ß-ionone could induce autophagy, as indicated by LC3 puncta in 786-O and ACHN cell lines and the expression of LC3 in ß-ionone-treated RCC cells. To further explore the underlying mechanism, we assessed liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) signaling pathway activity, and the results showed that ß-ionone inhibited the proliferation of RCC cells by inducing autophagy via the LKB1/AMPK signaling pathway. In summary, our findings provide a new therapeutic strategy of ß-ionone-induced autophagy in RCC.

Engineering a fermenting yeast able to produce the fragrant ß-ionone apocarotenoid for enhanced aroma properties in wine.

Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as ß-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing ß-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, ß-carotene. Haploid laboratory strains of S. cerevisiae were constructed with and without integrated carotenogenic genes and transformed with a plasmid containing the genes of CCD1. These strains were then mated with a sporulated diploid wine industry yeast, VIN13, and four resultant crosses-designated MQ01-MQ04-which were capable of fermenting the must to dryness were compared for their ability to release ß-ionone. Analyses of their fermentation products showed that the MQ01 strain produced a high level of ß-ionone and offers a fermenting hybrid yeast with the potential to enhance the organoleptic qualities of wine.

High-pressure treatment enhanced aromatic compound concentrations of melon juice and its mechanism.

Introduction: The flavor deterioration blocks the development of melon juice. Methods: The effects of ultra-high temperature (UHT) and high pressure (HP) treatments on the aromatic compound concentrations of melon juice and their mechanisms were explored with fresh juice as the control. Results: A total of 57 volatile compounds were identified by gas chromatography-tandem mass spectrometry analysis. ß-ionone was shown to be the major aromatic component of melon juice for the first time. The HP at 200 MPa for 20 min increased the total volatile concentration of melon juice by 1.54 and 3.77 times the control and UHT, respectively. Moreover, the sum concentration of a major aromatic component in the HP treatment was 1.49 and 5.94 times higher than that of the control and UHT, respectively. Discussion: The HP treatment raised the concentration of volatile and aromatic components of melon juice by reducing their surface tension.

Hydrosols of Veronica Species-Natural Source of Free Volatile Compounds with Potential Pharmacological Interest.

In this study, free volatile compounds (FVCs) were isolated from the water fractions (hydrosols) of 10 Croatian Veronica species obtained by hydrodistillation (HD) and microwave-assisted extraction (MAE). The use of different isolation techniques is important for the analysis of the influence of extraction conditions on the qualitative and quantitative composition of the isolated constituents. The composition of the hydrosols was analyzed using gas chromatography and mass spectrometry. The compounds ß-ionone and benzene acetaldehyde were detected in all 10 Veronica hydrosols studied. E-caryophyllene was also identified in all isolates except the MAE isolate of V. arvensis L. Caryophyllene oxide was isolated in all isolates apart from the HD isolate of V. catenata Pennell. (E)-ß-Damascenone is significantly present in all isolates except the MAE isolates of V. catanata and V. cymbalaria Bodard. In these two species, α-muurolol was identified in a high percentage. The same basic phytochemical constituents and compounds characteristic of a given Veronica species suggest the importance of further research. Antioxidant activity was tested for all extracts using two methods, ORAC and DPPH. Therefore, it is crucial to identify as many specialized metabolites from Veronica species as possible, especially hydrosols, which are natural products of potential pharmacological interest.

Characteristics of a new carotenoid cleavage dioxygenase NtCCD10 derived from Nicotiana tabacum.

MAIN CONCLUSION: A new carotenoid cleavage dioxygenase NtCCD10 from tobacco was characterized. There is some difference between NtCCD10 and CCD1 in structure. NtCCD10 can cleave the C5-C6 (C5'-C6') and C9-C10 (C9'-C10') double bonds of carotenoids and has high catalytic activity. Carotenoid cleavage dioxygenases (CCDs) cleave carotenoids to produce a variety of apocarotenoids, which have important biological functions for organisms in nature. There are eleven CCDs subfamilies in the plant kingdom, many of which have been extensively characterized in their functions. However, as a newly classified subfamily, the function of CCD10 has rarely been studied. In this work, the function of an NtCCD10 gene from dicotyledonous Nicotiana tabacum was cloned and characterized, and its phylogeny, molecular structural modeling and protein structure were also systematically analyzed. Like other CCDs, NtCCD10 also possesses a seven bladed ß-propeller with Fe2+ cofactor in its center constituting the active site of the enzyme. The Fe2+ is also coordinated bonding with four conserved histidine residues. Meanwhile, NtCCD10 also has many unique features, such as its α1 and α3 helixes are not anti-parallel, a special ß-sheet and a longer access tunnel for substrates. When expressed in engineered Escherichia coli (producing phytoene, lycopene, ß-carotene, and zeaxanthin) and Saccharomyces cerevisiae (producing ß-carotene), NtCCD10 could symmetrically cleave phytoene and ß-carotene at the C9-C10 and C9'-C10' positions to produce geranylacetone and ß-ionone, respectively. In addition, NtCCD10 could also cleave the C5-C6 and C5'-C6' double bonds of lycopene to generate 6-methyl-5-heptene-2-one (MHO). NtCCD10 has higher catalytic activity than PhCCD1 in yeast, which provides a good candidate CCD for biosynthesis of ß-ionone and has potential applications in biotechnological industry. This study identified the taxonomic position and catalytic activity of the first NtCCD10 in dicotyledonous plants. This will provide a reference for the discovery and functional identification of CCD10 enzymes in dicotyledons.

Characteristics of NtCCD1-3 from tobacco, and protein engineering of the CCD1 to enhance ß -ionone production in yeast.

Biosynthesis of ß-ionone by microbial cell factories has become a promising way to obtain natural ß-ionone. The catalytic activity of carotenoid cleavage dioxygenase 1 (CCD1) in cleavage of ß-carotene to ß-ionone severely limits its biosynthesis. In this study, NtCCD1-3 from Nicotiana tabacum with high ability to cleave ß-carotene was screened. Multiple strategies for improving the ß-ionone yield in Saccharomyces cerevisiae were performed. The results showed that NtCCD1-3 could cleave a variety of caroteniods at the 9,10 (9',10') double bonds and lycopene at the 5,6 (5',6') positions. The insertion site delta for NtCCD1-3 gene was more suitable for enhancing the yield of ß-ionone, showing 19.1-fold increase compared with the rox1 site. More importantly, mutant K38A of NtCCD1-3 in membrane-bonding domains could greatly promote ß-ionone production by more than 3-fold. We also found that overexpression of the NADH kinase Pos5 could improve ß-ionone yield up to 1.5 times. These results may provide valuable references for biosynthesis of ß-ionone.