Protein engineering of carotenoid cleavage dioxygenases to optimize ß-ionone biosynthesis in yeast cell factories.

Synthesis of ß-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a ß-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by ß-ionone quantification via RP-HPLC. Two single-point mutants increased ß-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed ß-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of ß-ionone synthesized by metabolically engineered yeast.

COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors.

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing ß-carotene and co-producing ß-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.

Efficient production of Pseudoionone with multipathway engineering in Escherichia coli.

AIMS: This study aimed to achieve efficient production of pseudoionone in Escherichia coli. METHODS AND RESULTS: Firstly, a stable lycopene-producing strain was constructed by integrating lycopene biosynthetic pathway into the chromosome. Further introduction of the Cucumis melo carotenoid cleavage dioxygenase 1 (cmCCD1) gene resulted in the production of pseudoionone. Then, the tricarboxylic acid cycle (i.e. sdhABCD and sucAB), pentose phosphate pathway (i.e. talB), and methylerythritol 4-phosphate pathway (i.e. idi and dxs) genes were modulated to improve the production of lycopene and accordingly pseudoionone. Regulation of the expression of talB and sdhABCD for improving NADPH supplies led to a 2·81-fold increase of pseudoionone production. Further engineering of dxs and idi increased the production of pseudoionone by 8·34-fold. Followed by bioprocess optimization, 24 mg l-1 pseudoionone with a specific production of 8·55 mg g-1 in shake flask was achieved. CONCLUSIONS: The combination of metabolic engineering and bioprocess engineering increased the production of pseudoionone by more than 185-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study promises a green and sustainable route for pseudoionone production using a microbial cell factory.

RNA-Seq analysis and comparison of the enzymes involved in ionone synthesis of three cultivars of Osmanthus.

To comprehend the molecular mechanisms that control the differences in the composition of Osmanthus essential oils, the RNA-seq data and differentially expressed genes in different cultivar Osmanthus were studied. cDNA libraries of "jinqiugui," "baijie," and "rixianggui" were sequenced using Illumina HiSeq TM 2000. All of the enzymes involved in ionone synthesis were verified. DEGs were revealed and their enriched pathways were analyzed. A total of 20 DEGsencoding four enzymes that were potential candidates involved in ionone biosynthesis, as well as ispH, GPPS, ZDS, and CCD. It provided a way for Osmanthus oil monomer material to be synthesized in vitro.

A "plug-n-play" modular metabolic system for the production of apocarotenoids.

Apocarotenoids, such as α-, ß-ionone, and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce ß-ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli-based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N-terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α-ionone production from 0.5 mg/L to 30 mg/L in flasks, producing 480 mg/L of α-ionone in fed-batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re-engineered to produce 32 mg/L and 500 mg/L of ß-ionone in flask and bioreactor, respectively (>80-fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the "plug-n-play" modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes.

Isolation and Functional Characterization of Carotenoid Cleavage Dioxygenase-1 from Laurus nobilis L. (Bay Laurel) Fruits.

Bay laurel (Laurus nobilis L.) is an agriculturally important tree used in food, drugs, and the cosmetics industry. Many of the health beneficial properties of bay laurel are due to volatile terpene metabolites that they contain, including various norisoprenoids. Despite their importance, little is known about the norisoprenoid biosynthesis in Laurus nobilis fruits. We found that the volatile norisoprenoids 6-methyl-5-hepten-2-one, pseudoionone, and ß-ionone accumulated in Laurus nobilis fruits in a pattern reflecting their carotenoid content. A full-length cDNA encoding a potential carotenoid cleavage dioxygenase (LnCCD1) was isolated. The LnCCD1 gene was overexpressed in Escherichia coli, and recombinant protein was assayed for its cleavage activity with an array of carotenoid substrates. The LnCCD1 protein was able to cleave a variety of carotenoids at the 9,10 (9',10') and 5,6 (5',6') positions to produce 6-methyl-5-hepten-2-one, pseudoionone, ß-ionone, and α-ionone. Our results suggest a role for LnCCD1 in Laurus nobilis fruit flavor biosynthesis.

Production of ß-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae.

BACKGROUND: Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants-like raspberries and roses-by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid ß-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP. RESULTS: Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of ß-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased ß-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of ß-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase. CONCLUSIONS: An efficient ß-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest ß-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.

Influence of viticulture practices on grape aroma precursors and their relation with wine aroma.

BACKGROUND: The effects of two different operations in the vineyard (basal leaf plucking and head trimming) on the synthesis of aromatic precursors in the grape and their impact on wine aroma have been studied and compared with a control sample. The study was carried out over two consecutive years with four different varieties. Glycosidic precursors were analysed in grapes and volatile compounds were studied in the wines. ANOVA studies were performed to study the effect of the vintage, variety and treatment for each of the compounds released from their precursors. RESULTS: Regarding treatment, the highest values in the concentration of free aroma compounds were achieved in the leaf plucking grapes, except for Chardonnay. Significant and positive correlations between aromatic precursors of terpenes present in grapes and their released form in wines were found for all varieties. For norisoprenoids, significant and positive correlations were exclusively found for Chardonnay and for phenols and vanillins in the year 2010 the correlations were high in three of the four varieties studied. CONCLUSION: In the assays of the 2 years, more precursors were synthesised in Merlot, Gewurztraminer and Tempranillo grapes if the vineyards were plucked.

Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene.

Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and ß-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and ß-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

Reaction specificities of the ε-ionone-forming lycopene cyclase from rice (Oryza sativa) elucidated in vitro.

Lycopene cyclases responsible for the formation of ε-ionone rings (LCYe) mark a plant-specific bifurcation of carotenogenesis. We investigated purified rice LCYe (OsLCYe) in a liposome-based biphasic assay system. OsLCYe depends on reduced flavin cofactors stabilizing a transient state formed during the non-redox cyclization reaction. In contrast to OsLCYb, OsLCYe produces predominantly monocyclic products and monocyclic carotene intermediates are not suitable substrates. Determination of the OsLCYe reaction specificities and the combined use of OsLCYb allow the characterization of the reaction sequence leading to heterocyclic carotenoids. It was also found that 5-cis-lycopene, which was thought to be decisive for ε-cyclization, was not involved in the reaction, with OsLCYe acting as an exclusion filter for this naturally occurring isomer.

Use of the plant Bellardia trixago for the enantiospecific synthesis of odorant products.

An easy procedure to obtain extracts enriched in trixagol monomalonylesther (1) from aerial parts of the plant Belladia trixago chemotype Trix was developed. Preparation of (+)-dihydro-gamma-ionone (4) was carried out directly from the extracts with good yields by selective oxidation. Other interesting odorant products as alpha-ambrinol (5), ambraldehyde (6) and the tricyclic compound 7 were synthesized very efficiently using (4) as intermediate.

Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in beta-ionone release.

Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound beta-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of beta-carotene and emission of beta-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.

Biosynthesis of monoterpenes and norisoprenoids in raspberry fruits (Rubus idaeus L.): the role of cytosolic mevalonate and plastidial methylerythritol phosphate pathway.

The biosynthesis of the monoterpenes (-)-alpha-pinene, linalool, and the norisoprenoids alpha- and beta-ionone in raspberry fruits (rubus idaeus L.) was investigated by in vivo feeding experiments with [5,5-(2)H2]-mevalonic acid lactone and [5,5-(2)H2]-1-deoxy-D-xylulose. The volatile compounds were extracted by stirbar sorptive extraction and analyzed using thermal desorption-multidimensional gas chromatography-mass spectrometry (TD-enantio-MDGC-MS). The feeding experiments demonstrate that (-)-alpha-pinene and (S)-linalool are exclusively synthesized via the cytosolic mevalonic acid pathway. In contrast, (2)H-labeled (R)-(E)-alpha-ionone and (2)H-labeled (E)-beta-ionone are detectable after application of d2-1-deoxy-D-xylulose and d2-mevalonic acid lactone, respectively. However, (R)-linalool reveals no incorporation of either one of the fed precursors, even though this enantiomer is detectable in the fruit tissue.

Influence of plant water status on the production of C13-norisoprenoid precursors in Vitis vinifera L. Cv. cabernet sauvignon grape berries.

The influence of irrigation strategy on grape berry carotenoids and C13-norisoprenoid precursors was investigated for Vitis vinifera L. cv. Cabernet Sauvignon. Two irrigation treatments were compared, one in which vines received reduced irrigation applied alternately to either side of the vine (partial rootzone drying, PRD) and a second control treatment in which water was applied to both sides of the vine. Over the two years of the experiments, PRD vines received on average 66% of the water applied to the controls. Initially, the PRD treatment did not alter midday leaf (psiL) and stem (psiS) water potential relative to the control, but decreased stomatal conductance (gs). Continued exposure to the PRD treatment resulted in treated grapevines experiencing hydraulic water deficit relative to the control treatment and induced lowered midday psiL and psiS, which was also reflected in decreased berry weight at harvest. In both irrigation treatments, the most abundant grape berry carotenoids, beta-carotene and lutein, followed the developmental pattern typical of other grape varieties, decreasing post-veraison. At certain points in time, as the fruit approached maturity, the concentration of these carotenoids was increased in fruit of PRD-treated vines relative to the controls. This effect was greater for lutein than for beta-carotene. PRD consistently caused increases in the concentration of hydrolytically released C13-norisoprenoids beta-damascenone, beta-ionone, and 1,1,6-trimethyl-1,2-dihydronaphthalene in fruit at harvest (24 degrees Brix) over two seasons. The effect of the PRD treatment on the concentration of hydrolytically released C13-norisoprenoids was greater in the second of the two seasons of the experiment and was also reflected in an increase in total C13-norisoprenoid content per berry. This suggests that the increases in the concentration of the C13-norisoprenoids in response to PRD were independent of water deficit induced changes in berry size and were not the result of an altered berry surface area to volume ratio.

The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles beta-ionone, pseudoionone, and geranylacetone.

Volatile terpenoid compounds, potentially derived from carotenoids, are important components of flavor and aroma in many fruits, vegetables and ornamentals. Despite their importance, little is known about the enzymes that generate these volatiles. The tomato genome contains two closely related genes potentially encoding carotenoid cleavage dioxygenases, LeCCD1A and LeCCD1B. A quantitative reverse transcriptase-polymerase chain reaction analysis revealed that one of these two genes, LeCCD1B, is highly expressed in ripening fruit (4 days post-breaker), where it constitutes 0.11% of total RNA. Unlike the related neoxanthin cleavage dioxygenases, import assays using pea chloroplasts showed that the LeCCD1 proteins are not plastid-localized. The biochemical functions of the LeCCD1 proteins were determined by bacterial expression and in vitro assays, where it was shown that they symmetrically cleave multiple carotenoid substrates at the 9,10 (9',10') positions to produce a C14 dialdehyde and two C13 cyclohexones that vary depending on the substrate. The potential roles of the LeCCD1 genes in vivo were assessed in transgenic tomato plants constitutively expressing the LeCCD1B gene in reverse orientation. This over-expression of the antisense transcript led to 87-93% reductions in mRNA levels of both LeCCD1A and LeCCD1B in the leaves and fruits of selected lines. Transgenic plants exhibited no obvious morphological alterations. High-performance liquid chromatography analysis showed no significant modification in the carotenoid content of fruit tissue. However, volatile analysis showed a > or =50% decrease in beta-ionone (a beta-carotene-derived C13 cyclohexone) and a > or =60% decrease in geranylacetone (a C13 acyclic product likely derived from a lycopene precursor) in selected lines, implicating the LeCCD1 genes in the formation of these important flavor volatiles in vivo.

Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission of beta-ionone, a fragrance volatile of petunia flowers.

Carotenoids are thought to be the precursors of terpenoid volatile compounds that contribute to flavor and aroma. One such volatile, beta-ionone, is important to fragrance in many flowers, including petunia (Petunia hybrida). However, little is known about the factors regulating its synthesis in vivo. The petunia genome contains a gene encoding a 9,10(9',10') carotenoid cleavage dioxygenase, PhCCD1. The PhCCD1 is 94% identical to LeCCD1A, an enzyme responsible for formation of beta-ionone in tomato (Lycopersicon esculentum; Simkin AJ, Schwartz SH, Auldridge M, Taylor MG, Klee HJ [2004] Plant J [in press]). Reduction of PhCCD1 transcript levels in transgenic plants led to a 58% to 76% decrease in beta-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis. Quantitative reverse transcription-PCR analysis revealed that PhCCD1 is highly expressed in corollas and leaves, where it constitutes approximately 0.04% and 0.02% of total RNA, respectively. PhCCD1 is light-inducible and exhibits a circadian rhythm in both leaves and flowers. beta-Ionone emission by flowers occurred principally during daylight hours, paralleling PhCCD1 expression in corollas. The results indicate that PhCCD1 activity and beta-ionone emission are likely regulated at the level of transcript.