Production of alpha-(R)-(E)-(+)-ionone in recombinant Saccharomyces cerevisiae

Abstract

This invention provides improved biological synthesis of the apocarotenoid α-ionone in Saccharomyces cerevisiae. The final native step involved in the natural apocarotenoid pathway depends on an endogenous farnesyl pyrophosphate synthase (FPPs). From there, heterologous geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtl), lycopene ε-cyclase (LycE) and a Carotenoid Cleavage Dioxygenase (CCD1) are required to complete the synthesis of α-ionone. Lycopene ε-cyclase from lettuce (Lactuca sativa) or modified cyclase from Arabidopsis thaliana was used to overproduce lycopene which was then cleaved by the carotenoid cleavage dioxygenase from Petunia hybrida (Ph-CCD1).

Claims

1. A method of producing enantiomerically pure (R)-(E)-(+)-alpha-ionone in Saccharomyces cervisiae comprising the steps of: (a) constructing recombinant yeast cells that overexpress native nucleic acids, or modified versions thereof, encoding at least one enzyme of the mevalonate pathway for synthesizing farnesyl pyrophosphate, wherein expression of the one or more enzymes is under control of constitutive or inducible promoters; (b) modifying the recombinant yeast cells from step (a) to further comprise heterologous nucleic acids that encode enzymes of an apocarotenoid pathway for synthesizing alpha-ionone, wherein said apocarotenoid pathway includes (i) an enzyme that condenses farnesyl pyrophosphate and isopentenyl pyrophosphate to form geranylgeranyl pyrophosphate, (ii) an enzyme that condenses two molecules of geranylgeranyl pyrophosphate to form phytoene, (iii) an enzyme that converts phytoene to lycopene, (iv) an enzyme that cyclizes lycopene to form δ-carotene and/or ε-carotene, and (v) an enzyme that cleaves δ-carotene and/or ε-carotene to produce alpha-ionone.

2. The method of claim 1, wherein the yeast cells are selected from the group consisting of Pichia pastoris, Yarrowia lipolytica, and Saccharomyces cerevisiae.

3. The method of claim 1, wherein the enzyme that condenses farnesyl pyrophosphate with isopentenyl pyrophosphate is a geranylgeranyl pyrophosphate synthase (CrtE).

4. The method of claim 3, wherein the geranylgeranyl pyrophosphate synthase (CrtE) is from Xanthophyllomyces dendrorhous.

5. The method of claim 1, wherein the enzyme that cyclizes lycopene is lycopene ε-cyclase (LcyE) from one or more of the following: Zea mays, Chromochloris zofingiensis, Marchantia polymorpha and Lactuca sativa.

6. The method of claim 1, wherein the enzyme that cleaves δ-carotene and/or ε-carotene is an oxidoreductase.

7. The method of claim 6, wherein the oxioreductase is selected from the group consisting of monooxygenase, dioxygenase, and peroxidase.

8. The method of claim 7, wherein the monooxygenase and dioxygenase are CCDs.

9. The method of claim 6, wherein the oxioreductase is a CCD1 from Petunia hybrida, Osmanthus fragrans, Arabidopsis thaliana, or Vitis vinifera.

10. The method of claim 1, wherein the nucleic acids are expressed using yeast centromeric plasmids, high copy number plasmids or integration plasmids, and integrated into specific and stable yeast genomic sites.

11. The method of claim 1, wherein the alpha-ionone is produced in an amount greater than 1 mg per gram of dry cell weight.

12. The method of claim 11, wherein the alpha-ionone is produced in less than 100 hours.

13. The method of claim 1, wherein the recombinant yeast cells are transformed to include nucleic acids having the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:10.

14. The method of claim 1, wherein the recombinant yeast cells are transformed to include nucleic acids having the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:10.

Description

DESCRIPTION OF THE FIGURES

[0018] FIG. 1 shows the chemical structures of (R)-(E)-(+)-alpha-ionone and (S)-(E)-(−)-alpha-ionone;

[0019] FIG. 2 shows a heterologous pathway for the synthesis of α-ionone;

[0020] FIG. 3 is the amino acid sequence of geranylgeranyl pyrophosphate synthase from Xanthophyllomyces dendrorhous (Xd-crtE) (SEQ ID NO:1);

[0021] FIG. 4 is the amino acid sequence of geranylgeranyl pyrophosphate synthase phytoene synthase from Pantoea agglomerans (Pa-crtB) (SEQ ID NO:2);

[0022] FIG. 5 is the amino acid sequence of lycopene ε-cyclase from Zea mays (Zm-LycE) (SEQ ID NO:3);

[0023] FIG. 6 is the amino acid sequence of phytoene desaturase from Xanthophyllomyces dendrorhous (Xd-crtl) (SEQ ID NO:4);

[0024] FIG. 7 is the amino acid sequence of lycopene ε-cyclase from Zea mays (Zm-LycE) (SEQ ID NO:5);

[0025] FIG. 8 is the amino acid sequence of L461H mutant lycopene ε-cyclase from Zea mays (Zm-LycE) (SEQ ID NO:6);

[0026] FIG. 9 is the amino acid sequence of lycopene ε-cyclase from Lactuca sativa

[0027] (Ls-LycE) (SEQ ID NO:7);

[0028] FIG. 10 is the amino acid sequence of lycopene ε-cyclase from green alga Chlorella zofingiensis (Cz-LycE) (SEQ ID NO:8);

[0029] FIG. 11 is the amino acid sequence of lycopene ε-cyclase from the liverwort Marchantia polymorpha (Mp-LycE) (SEQ ID NO:9);

[0030] FIG. 12 is the amino acid sequence of carotenoid cleavage dioxygenase from Petunia hybrida (Ph-CCD1) (SEQ ID NO:10);

[0031] FIG. 13 is a plasmid map of an XI-5 divergent vector containing the crtE and crtB genes;

[0032] FIG. 14 is a plasmid map of an XI-5 tandem vector containing the crtE and crtB genes;

[0033] FIG. 15 is a plasmid map of an XI-3 divergent vector containing the crtl and lycE genes;

[0034] FIG. 16 is a plasmid map of an XI-3 tandem vector containing the crtl and lycE genes;

[0035] FIG. 17 is a plasmid map of an X-2 vector containing the ccd1 gene;

[0036] FIG. 18 shows the specific α-ionone yield in mg/g of dry cell weight (DCW), as well as the residual carotenoidsm, of the characterized strains; and

[0037] FIG. 19 shows the carotenoid profiles of αε-1 and αε-3 strains.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventors of carrying out their invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide an improved method for producing α-ionone.

[0039] Alpha-ionone belongs to the C.sub.13-apocarotenoid or C.sub.13-norisoprenoid class of compounds. Apocarotenoids are natural aromatic compounds produced in green land plants by the enzymatic cleavage of carotenoids. Carotenes are forty-carbon molecules (C.sub.40) whose biosynthesis depends on the availability of two C.sub.5 universal isoprene-building units: isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Isoprene biosynthesis can be carried out through the mevalonate pathway (MVA) or, alternatively, by the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway. Because MVA pathway is naturally present in S. cerevisiae, the present invention uses this organism for carotene biosynthesis. The last native step involved in the natural apocarotenoid pathway depends on an endogenous farnesyl pyrophosphate synthase (FPPs). From there, heterologous geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (ctrl), lycopene ε-cyclase (LycE) and a Carotenoid Cleavage Dioxygenase (CCD1) are required to complete the synthesis of α-ionone.

[0040] Lycopene ε-cyclase can produce either monocyclic δ-carotene or dicyclic ε-carotene. Most green land plants such as Arabidopsis thaliana or Solanum lycopersicum have lycopene ε-cyclases with preferential monocyclic activity that yields δ-carotene as the main product. However, lycopene ε-cyclase from lettuce (Lactuca sativa) performs an effective double cyclization of lycopene to produce primarily ε-carotene. Using modified proteins and site-specific mutations (single amino acid mutation of leucine to histidine) gives A. thaliana LycE double cyclization activity similar to lycopene ε-cyclases from L. sativa (see reference 4) in E. coli cells. The impact of this single amino acid change was also validated in an L461H LycE mutant from Zea mays in E. coli cells. Although both δ- and ε-carotenes could be substrates for CCD1 cleavage for α-ionone production, ε-carotene provides better yields because the stoichiometry produces two molecules of α-ionone per molecule of ε-carotene.

[0041] Genes that encode carotenogenic and carotenoid cleavage enzymes are described below with sequences shown in FIGS. 3-12. Some of these sequences have been codon optimized for its expression in S. cerevisiae. The screening of five different lycopene ε-cyclase candidates has made it possible to find the most effective sequence for the yeast platform.

[0042] Lycopene ε-cyclase can produce either monocyclic δ-carotene or dicyclic ε-carotene. Most green land plants such as Arabidopsis thaliana or Solanum lycopersicum have lycopene

[0043] Geranylgeranyl pyrophosphate synthase from Xanthophyllomyces dendrorhous (Xd-crtE); phytoene synthase from Pantoea agglomerans (Pa-crtB) or the mutant bifunctional phytoene synthase/lycopene cyclase from Xanthophyllomyces dendrorhous (Xd-crtYB*) are described by reference 10 as producing a tetraterpene (C.sub.40) overproducing Saccharomyces cerevisiae that accumulated up to 1.61 g/L of lycopene in a fed-batch glucose fermentation.

[0044] Phytoene desaturase from Blakeslea trispora (Bt-crtl) or Phytoene desaturase from Xanthophyllomyces dendrorhous were used by Chen et al., 2016 (reference 2) for host and pathway engineering in Saccharomyces cerevisiae to overproduce lycopene, thereby obtaining up to 1.64 g/L of this tetraterpene. Pathway engineering included enzyme screening of different phytoene desaturases (crtl). Chen et al., 2016 showed that regardless of the combination of crtB and crtE genes, Blakeslea trispora crtl increased lycopene production with respect to homologues from other organisms.

[0045] Lycopene ε-cyclase from Lactuca sativa (Ls-LycE): The only reported native ε-cyclase that has preferred lycopene bi-cyclase over mono-cyclase activity, confirmed by expression in lycopene-producing strain of Escherichia coli (see reference 4).

[0046] Native and L461H mutated lycopene ε-cyclase from Zea mays (Zm-LycE): Bai et al., 2009 (reference 1) isolated and expressed in E. coli a maize lycopene ε-cyclase that produced preferentially δ-carotene. Bai et al., 2009 showed that a directed gene mutagenesis of single amino acid (L461H) was sufficient to allow this enzyme to produce mainly ε-carotene.

[0047] Lycopene ε-cyclase from the green alga Chlorella zofingiensis (Cz-LycE): coding for an enzyme with proven mono-cyclase activity to yield δ-carotene when it is expressed in lycopene-accumulating strain of E. coli (reference 3). This gene has approximately 40% of sequence identity with LycE from green land plants (GLP) and represents a phylogenetic alternative to Z. mays or L. sativa cyclase genes.

[0048] Lycopene ε-cyclase from liverwort Marchantia polymorpha (Mp-LycE): liverworts are known to be early land plants, phylogenetically are between green algae and higher GLP. Expression of LycE from M. polymorpha produces similar amounts of δ-carotene and ε-carotene.

[0049] Carotenoid cleavage dioxygenase from Petunia hybrida (Ph-CCD1): to obtain final α-ionone strain(s) it is necessary to express a CCD1 gene to cleave δ and/or ε-carotene. CCD1 enzymes cleavage substrates include either β- or ε-ring carotenoids (see reference 6).

[0050] Vectors and Cloning.

[0051] To reconstruct the α-ionone pathway in S. cerevisiae it is necessary to clone the carotenogenic genes into suitable expression vectors. The most commonly used plasmids in yeast are integrative and episomal vectors. Although an increase in the number of gene copies can be seen by using episomal vectors, gene integration is preferable due to the resulting higher strain stability over time. In order to perform vector integration, some sequence modules are required for the DNA recombination, strain selection, marker recycling and gene expression. The present invention uses a collection of vectors disclosed in reference 7 without including the marker genes. These plasmids allow integration and expression in a divergent mode of two or more genes per each vector due to the presence of a bidirectional fused promoter, or in a tandem mode, separating the genes by different promoters. Two or more different transcription terminator sequences are placed downstream of each gene, thereby preventing unwanted recombination events. These cloning vectors also contain E. coli replication origin and ampicillin resistance sites in order to enable easy amplification of these vectors to obtain sufficient amounts of DNA to transform yeast. Depending on their homologous regions (UP and DOWN), vectors can be integrated in several specific sites of chromosomes X, XI and XII of S. cerevisiae by means of a double crossover mechanism. These sites were selected based on their stability, expression levels and growth impact on yeast. FIGS. 13-17 show the minimum set of integrating plasmids needed to construct α-ionone-producing strains of S. cerevisiae.

[0052] These vectors can be constructed by using different molecular biology techniques: including restriction cloning or PCR-based seamless cloning such as Gibson Assembly or Golden Gate Assembly.

[0053] Transformation and Selection of Strains.

[0054] Transformation of yeast with integrative vectors can be performed according to general protocols for LiAc/SS carrier DNA/PEG method (see reference 5). Preferably, plasmids are digested with Swal/Smil enzyme to linearize the DNA and increase transformation efficiency.

[0055] Selection of transformants was performed by plate-color screening and colony PCR. Even though genomic recombination of integrative vectors is much less probable than the simple incorporation event of episomal vector, using 1-5 μg of plasmid per transformation is sufficient to obtain several colonies. Depending on parental strain, carotenogenic transformants could show some phenotypic variability. This is noticeable in the diversity of colony colors obtained in one round of transformation. Visual screening can be useful to isolate different phenotypes for characterization. Usually differences in color between two colonies imply variations in carotenoid levels and/or carotenoid composition. PCR and genomic sequencing can then be used to check that those vectors are correctly integrated on the specific genomic sites.

[0056] Strain Characterization: Carotenoid and C.sub.13-Apocarotenoid Analysis.

[0057] Characterization of the generated strains is required for selecting candidates with the best productive potential. Shake flask cultures containing YPD (yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L) medium were used and carotenoids and aroma profiles were studied for each new strain. Carotenoids were extracted in hexane from 48-hour flask cultures. The resulting organic phase was directly analyzed by reverse phase HPLC. Carotenoids quantification in strains allows identification of possible bottlenecks in the pathway. For C.sub.13-apocarotenoid quantification, including α-ionone, supernatants obtained from 72-hour cultures were extracted with 10% (v/v) dodecane and analyzed by GC-MS.

Example: Construction and Characterization of α-Ionone-Producing Strains

[0058] Codon optimized gene synthesis was contracted to Genscript and received as pUC57 vectors. Genes and backbone of the expression vectors were amplified through the Phusion polymerase enzyme (NEB), using primers that contain homologous sequences for Gibson Assembly. PCR products were separated by electrophoresis and extracted by using a Promega purification kit. Purified PCR products were mixed with Gibson Assembly mix (New England Biolabs) and incubated for two hours at 50° C. Gibson reaction products were transformed in competent cells (E. coli TOP10, Thermo Scientific) and plated in LBA (LB medium with ampicillin). Screening of transformants was done by colony PCR and positive colonies were grown in LBA to purify plasmids by using miniprep kits (Promega). Correct assembly of vectors was checked by sequencing (Macrogen).

[0059] Assembled vectors were digested with Swal/Smil (Thermo Scientific) for three hours at 37° C. and 2 μg of digestion products were used directly for S. cerevisiae transformation. Yeast transformation was performed as described in the LiAc/SS carrier DNA/PEG method (for example see reference 5), and plated on YPD medium. After 10-15 transformant colonies were re-plated, PCR was used to confirm whether vectors were correctly inserted at specific genomic sites. Genomic DNAs were isolated with a purification kit (Promega) and final strains were confirmed by sequencing (Macrogen). As mentioned above, to construct α-ionone producing strains it is necessary to transform with a minimum of five genes that can be grouped preferably in two vectors and co-transformed simultaneously.

[0060] For quantitative carotenoid profiling, 2-5 mL of each culture was centrifuged, and pellets were washed with water. The pelleted cells were disrupted in 1 mL of hexane using 500 μL of glass beads to extract the carotenoids. Extracts were analyzed by reverse phase HPLC. Chromatography was carried on a 018 column, at 0.5 mL/min and 45° C., using a mobile phase composed of 85% acetonitrile, 10% methanol and 5% isopropanol. Lycopene, δ-carotene, ε-carotene, and β-carotene were then quantified (see FIG. 19).

[0061] For GC-MS identification and quantification of α-ionone, a 20 μL sample of the organic phase was pipetted into a 20 mL headspace vial and diluted with 5 mL of a 30% (w/v) aqueous sodium chloride solution. For quantification, 50 μL of a 1-ppm ethanolic solution of β-damascenone-d4 was added to the vial. Automated headspace solid-phase micro-extraction HS-SPME was performed with a TriPlusRSH sampler robot.

[0062] Separation, identification and quantification of C.sub.13-apocarotenoids (β-ionone, α-ionone, geranyl-acetone, pseudo-ionone) were carried out by gas chromatography-mass spectrometry (SHIMADZU GCMS-QP2010) using a DB-WAXetr column (0.25 μm, 30 m×0.25 ID).

[0063] Table 1 (below) shows the α-ionone concentrations detected in dodecane extracts of 72-hr. cultures in mg/L of dodecane extracted and the mg of α-ionone per mg of cell dry weight. Note that strain αε-1 is more productive than strain αε-3.

TABLE-US-00001 TABLE 1 Characterization of alpha-ionone producing strains of Saccharomyces cerevisiae (CEN.PK based). alpha-ionone in alpha- dodecane layer ionone Strain Genotype OD600 (mg/L) (mg/g) αε-1 Xd-crtYB*, Xd-crtE, Xd-crtl, 20 90 1.07 Ls-LycE. Ph-CCD1 αε-3 Xd-crtYB*, Xd-crtE, Xd-crtl, 15 52 0.86 Ls-LycE, Ph-CCD1, Zm- LcyE

[0064] FIG. 18 summarizes the α-ionone yield in mg/g dry cell weight (DCW) and residual carotenoids after 72 hours cultivation in shake flasks. Table 1 indicates that αε-1 strain is a more suitable construct for production of α-ionone because it has greater ionone content, specific target production and room for improvements (δ-carotene and ε-carotene content is shown in FIG. 19).

[0065] Lycopene is the main carotenoid present in the cells and its accumulation appears to be detrimental for yeast growth. An increased efficiency of the LcyE (cyclase) and CDD1 activities are crucial for both detoxification and higher α-ionone production.

[0066] The following claims are thus to be understood to include what is specifically illustrated and described above, and what can be obviously substituted. Those skilled in the art will appreciate that various adaptations and modifications of the just-described preferred embodiment can be configured without departing from the scope of the invention. The illustrated embodiment has been set forth only for the purposes of example and should not be taken as limiting the invention.

REFERENCES

[0067] [1] Bai, L., Kim, E. H., Dellapenna, D., Brutnell, T. P. (2009) Novel lycopene epsilon cyclase activities in maize revealed through perturbation of carotenoid biosynthesis. Plant J 59:588-599. [0068] [2] Chen, Y., Xiao, W., Wang, Y., Liu, H., Li, X., Yuan, Y. (2016) Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering. Microb Cell Fact 15:113. [0069] [3] Cordero, B. F., Couso, I., Leon, R., Rodriguez, H., Vargas, M. A. (2012) Isolation and characterization of a lycopene ε-Cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light. Mar Drugs 10:2069-2088. [0070] [4] Cunningham, F. X., Gantt, E. (2001) One ring or two? Determination of ring number in carotenoids by lycopene ε-cyclases. Proc Natl Acad Sci USA 98:2905-2910. [0071] [5] Gietz, R. D. (2014) Yeast transformation by the LiAc/SS carrier DNA/PEG method. In: Yeast Protocols, 3rd edn. Humana Press, New York. [0072] [6] Huang, F. C., Horvath, G., Molnár, P., Turcsi, E., Deli, J., Schrader, J., Sandmann, G., Schmidt, H., Schwab, W. (2009) Substrate promiscuity of RdCCD1, a carotenoid cleavage oxygenase from Rosa damascena. Phytochemistry 70:457-464. [0073] [7] Mikkelsen, M D, Buron, L D, Salomonsen, B., Olsen, C. E., Hansen, B. G., Mortensen, U. H., Halkier, B. A. (2012) Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab Eng 14:104-111. [0074] [8] Werkhoff, P., Bretschneider, W., Güntert, M., Hopp, R., Surburg, H. (1991) Chirospecific analysis in flavor and essential oil chemistry part B. Direct enantiomer resolution of trans-α-ionone and trans-α-damascone by inclusion gas chromatography. Eur Food Res Technol 192:111-115. [0075] [9] Wang, C., Liwei, M., Park, J. B., Jeong, S. H., Wei, G., Wang, Y., Kim, S. W. (2018) Microbial Platform for Terpenoid Production: Escherichia coli and Yeast. Front. Microbiol. 9:2460 [0076] [10] Xie, W., Lv, X., Ye, L., Zhou, P., Yu, H. (2015) Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metab Eng 30:69-78.