A METHOD FOR THE PRODUCTION OF PLANTS WITH ALTERED PHOTORESPIRATION AND IMPROVED CO2 FIXATION
20220315942 · 2022-10-06
Assignee
- Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften e.V. (Munich, DE)
- Heinrich-Heine-Universität Düsseldorf (Düsseldorf, DE)
Inventors
- Lennart Schada Von Borzyskowski (Marburg, DE)
- Tobias Jürgen Erb (Marburg, DE)
- Andreas Paul Michael Weber (Düsseldorf, DE)
- Marc-Sven Roll (Düsseldorf, DE)
Cpc classification
C12N15/8245
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to transgenic plants with altered photorespiration and improved CO.sub.2 fixation as well as a method of producing said transgenic plants. Particularly, the transgenic plants show an improved growth rate, productivity and energy conversion efficiency. This method can be successfully applied to many agricultural crop plants with nutritional and medicinal uses.
Claims
1. A method for the production of a transgenic plant with altered photorespiration and improved CO.sub.2 fixation, comprising introducing into a cell or tissue of said plant one or more nucleic acids encoding at least four polypeptides having the enzymatic activities of (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14, (b) erythro-β-hydroxyaspartate dehydratase belonging to the EC class 4.3.1.20, (c) iminosuccinate reductase and (d) aspartate-glyoxylate transaminase, wherein the introduction of said nucleic acid(s) results in a de novo expression of the at least four polypeptides having the enzymatic activities of (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14, (b) erythro-β-hydroxyaspartate dehydratase belonging to the EC class 4.3.1.20, (c) iminosuccinate reductase and (d) aspartate-glyoxylate transaminase, wherein the polypeptide having the enzymatic activity of (c) iminosuccinate reductase comprises an amino acid sequence selected from SEQ ID NO: 1-299, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-299; and the polypeptide having the enzymatic activity of (d) aspartate-glyoxylate transaminase comprises an amino acid sequence selected from SEQ ID NO: 300-599, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 300-599.
2. The method of claim 1, wherein the polypeptide having the enzymatic activity of (c) iminosuccinate reductase comprises an amino acid sequence selected from SEQ ID NO: 1, 7, 22, 25, 26, 39, 47, 58, 75, 123, 135, and 160, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, 7, 22, 25, 26, 39, 47, 58, 75, 123, 135, and 160.
3. The method of claim 1, wherein the polypeptide having the enzymatic activity of (c) iminosuccinate reductase comprises an amino acid sequence as set forth in SEQ ID NO: 135, or an amino acid sequence having at least 80% sequence identity to said sequence; and the polypeptide having the enzymatic activity of (d) aspartate-glyoxylate transaminase comprises an amino acid sequence as set forth in SEQ ID NO: 433, or an amino acid sequence having at least 80% sequence identity to said sequence.
4. The method of claim 1, wherein the polypeptides having the enzymatic activities (a)-(d) comprise an amino acid sequence targeting said polypeptides to the peroxisomes.
5. The method of claim 4, wherein the (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14 is C-terminally fused to a peroxisomal targeting signal 2 of SEQ ID NO: 952 and the polypeptides having the enzymatic activities (b)-(d) are N-terminally fused to a peroxisomal targeting signal 1 of amino acid sequence SKL.
6. The method of claim 1, wherein the one or more nucleic acids further encode a polypeptide having the enzymatic activity of (e) glycolate dehydrogenase belonging to the EC class 1.1.99.14, wherein the introduction of said nucleic acid(s) results in a de novo expression of at least five polypeptides having the enzymatic activity of (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14, (b) erythro-β-hydroxyaspartate dehydratase belonging to the EC class 4.3.1.20, (c) iminosuccinate reductase, (d) aspartate-glyoxylate transaminase, and (e) glycolate dehydrogenase belonging to the EC class 1.1.99.14, wherein said polypeptides having the enzymatic activities (a)-(e) are localized in cellular mitochondria, and wherein said polypeptides having the enzymatic activities (a)-(e) are N-terminally fused to a serine hydroxymethyltransferase 1 target peptide of SEQ ID NO: 919.
7. The method of claim 1, wherein the one or more nucleic acids further encode two polypeptides having the enzymatic activities of (e) glycolate dehydrogenase belonging to the EC class 1.1.99.14, (f) phosphoenolpyruvate carboxykinase belonging to the EC class 4.1.1.49 wherein the introduction of said nucleic acid(s) results in a de novo expression of at least six polypeptides having the enzymatic activities of (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14, (b) erythro-β-hydroxyaspartate dehydratase belonging to the EC class 4.3.1.20, (c) iminosuccinate reductase, (d) aspartate-glyoxylate transaminase, (e) glycolate dehydrogenase belonging to the EC class 1.1.99.14, and (f) phosphoenolpyruvate carboxykinase belonging to the EC class 4.1.1.49, wherein said polypeptides having the enzymatic activities (a)-(f) are localized in cellular chloroplasts, and wherein said polypeptides having the enzymatic activities (a)-(f) are N-terminally fused to Arabidopsis Ferredoxin-2 chloroplastic target peptide of SEQ ID NO: 917.
8. A transgenic plant comprising one or more heterologous nucleic acids encoding at least four polypeptides having the enzymatic activities of (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14, (b) erythro-β-hydroxyaspartate dehydratase belonging to the EC class 4.3.1.20, (c) iminosuccinate reductase comprising an amino acid sequence selected from SEQ ID NO: 1-299, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-299; and (d) aspartate-glyoxylate transaminase comprising an amino acid sequence selected from SEQ ID NO: 300-599, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 300-599.
9. The transgenic plant of claim 8, wherein the polypeptide having the enzymatic activity of (c) iminosuccinate reductase comprises an amino acid sequence selected from SEQ ID NO: 1, 7, 22, 25, 26, 39, 47, 58, 75, 123, 135, and 160, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, 7, 22, 25, 26, 39, 47, 58, 75, 123, 135, and 160.
10. The transgenic plant of claim 8, wherein the wherein the polypeptide having the enzymatic activity of (c) iminosuccinate reductase comprises an amino acid sequence as set forth in SEQ ID NO: 135, or an amino acid sequence having at least 80% sequence identity to said sequence; and the polypeptide having the enzymatic activity of (d) aspartate-glyoxylate transaminase comprises an amino acid sequence as set forth in SEQ ID NO: 433, or an amino acid sequence having at least 80% sequence identity to said sequence.
11. The transgenic plant of claim 8, wherein the (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14 is C-terminally fused to a peroxisomal targeting signal 2 of SEQ ID NO: 952 and the polypeptides having the enzymatic activities (b)-(d) are N-terminally fused to a peroxisomal targeting signal 1 of amino acid sequence SKL.
12. The transgenic plant of claim 8, wherein the one or more nucleic acids further encode a polypeptide having the enzymatic activity of (e) glycolate dehydrogenase belonging to the EC class 1.1.99.14, wherein said polypeptides having the enzymatic activities (a)-(e) are localized in cellular mitochondria, and wherein said polypeptides having the enzymatic activity (a)-(e) are N-terminally fused to a serine hydroxymethyltransferase 1 target peptide of SEQ ID NO: 919.
13. The transgenic plant of claim 8, wherein the one or more nucleic acids further encode two polypeptides having the enzymatic activities of (e) glycolate dehydrogenase belonging to the EC class 1.1.99.14, (f) phosphoenolpyruvate carboxykinase belonging to the EC class 4.1.1.49, wherein said polypeptides having the enzymatic activities (a)-(f) are localized in cellular chloroplasts, and wherein said polypeptides having the enzymatic activity (a)-(f) are N-terminally fused to Arabidopsis Ferredoxin-2 chloroplastic target peptide of SEQ ID NO: 917.
14. The transgenic plant of claim 8, wherein the plant is selected from Helianthus annuus, Brassica napus, Camelina sativa, Oryza sativa, Hordeum vulgare, Triticum spp., Avena sativa, Solanum lycopersicum, Solanum tuberosum, Glycine max, Beta vulgaris, Nicotiana tabacum, and Arabidopsis thaliana.
15. A nucleic acid construct comprising nucleic acid sequences encoding at least four polypeptides having the enzymatic activities of (a) erythro-β-hydroxyaspartate aldolase belonging to the EC class 4.1.3.14, (b) erythro-β-hydroxyaspartate dehydratase belonging to the EC class 4.3.1.20, (c) iminosuccinate reductase comprising an amino acid sequence selected from SEQ ID NO: 1-299, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-299; (d) aspartate-glyoxylate transaminase comprising an amino acid sequence selected from SEQ ID NO: 300-599, or an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 300-599, a selection marker nucleic acid, wherein the nucleic acid sequences are operably linked to at least one promoter for expression in a plant and are operably linked to at least one terminator.
Description
DESCRIPTION OF THE FIGURES
[0622]
[0623]
[0624]
[0625]
[0626]
[0627]
[0628]
[0629]
[0630]
[0631]
[0632]
[0633]
[0634]
[0635]
[0636]
[0637]
[0638]
[0639]
[0640]
[0641]
[0642]
[0643]
[0644]
[0645] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
[0646] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
EXAMPLES
Abbreviations and Acronyms
[0647] AsGAT, AGAT aspartate glyoxylate aminotransferase [0648] ISRed, ISR iminosuccinate reductase [0649] BHA erythro-β-hydroxyaspartate [0650] BHAA β-hydroxyaspartate aldolase [0651] BHAD β-hydroxyaspartate dehydratase [0652] BHAP β-hydroxyaspartate pathway [0653] CBB Calvin-Benson-Bassham [0654] DNA desoxyribo nucleic acid [0655] fwd forward primer [0656] FDH formate dehydrogenase [0657] MDH malate dehydrogenase [0658] rev reverse primer
Chemicals & Reagents
[0659] Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich and were of the highest purity available. Gene synthesis was also performed by Sigma-Aldrich.
Methods
[0660] Construction of expression vectors for heterologous expression of the enzymes ISRed, BHAA, BHAD and AsGAT as well as the regulatory protein BHAR The gene encoding for the iminosuccinate reductase enzyme from Paracoccus denitrificans DSM 413 (ISRed; nucleic acid sequence shown in SEQ ID NO: 969; amino acid sequence shown in SEQ ID NO: 135) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the ISRed gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers
TABLE-US-00003 (SEQ ID NO: 976) 5′-GACGCCTCATATGCTCGTCGTCGCCGAAAAG-3′ (SEQ ID NO: 977) 5′-GCCACTCCTCGAGTCAGATCTCGACCTCTTG-3′
[0661] The resulting PCR product was digested with the endonucleases NdeI and XhoI and ligated into the expression vector pET16b to create a vector for heterologous expression of ISRed.
[0662] The gene encoding for the β-hydroxyaspartate aldolase enzyme from Paracoccus denitrificans DSM 413 (BHAA; nucleic acid sequence shown in SEQ ID NO: 970; amino acid sequence shown in SEQ ID NO: 971) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the BHAA gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers
TABLE-US-00004 (SEQ ID NO: 978) 5′-GACGCCGCATATGAACGCGAAAACGGATTTC-3′ (SEQ ID NO: 979) 5′-GACACCTGGATCCTCAGTAGCCCTTTCCG-3′
[0663] The resulting PCR product was digested with the endonucleases NdeI and BamHI and ligated into the expression vector pET16b to create a vector for heterologous expression of BHAA.
[0664] The gene encoding for the β-hydroxyaspartate dehydratase enzyme from Paracoccus denitrificans DSM 413 (BHAD; nucleic acid sequence shown in SEQ ID NO: 972; amino acid sequence shown in SEQ ID NO: 973) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore).
[0665] To this end, the BHAD gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers
TABLE-US-00005 (SEQ ID NO: 980) 5′-GACGCTGCATATGTATATCCCGACCTATGAG-3′ (SEQ ID NO: 981) 5′-GACACTCGGATCCTCAGTTCCACGGCAGCTTG-3′
[0666] The resulting PCR product was digested with the endonucleases NdeI and BamHI and ligated into the expression vector pET16b to create a vector for heterologous expression of BHAD.
[0667] The gene encoding for the aspartate-glyoxylate aminotransferase enzyme from Paracoccus denitrificans DSM 413 (AsGAT; nucleic acid sequence shown in SEQ ID NO: 433; amino acid sequence shown in SEQ ID NO: 974) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the AsGAT gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers
TABLE-US-00006 (SEQ ID NO: 982) 5′-GCCACTACATATGACCAGCCAGAACCC-3′ (SEQ ID NO: 983) 5′-GCCACTCGGATCCTCAGGCGGCTTTCTTCTGC-3′
[0668] The resulting PCR product was digested with the endonucleases NdeI and BamHI and ligated into the expression vector pET16b to create a vector for heterologous expression of AsGAT.
[0669] The gene encoding for the BHA-regulatory protein from Paracoccus denitrificans DSM 413 (BHAR; nucleic acid sequence shown in SEQ ID NO: 975; amino acid sequence shown in SEQ ID NO: 732) was cloned into the standard expression vector pET16b (commercially available from Merck Millipore). To this end, the BHAR gene was amplified from genomic DNA of Paracoccus denitrificans DSM 413 with the primers
TABLE-US-00007 (SEQ ID NO: 984) 5′-GCCACATCATATGTCGGTTCAAATCC-3′ (SEQ ID NO: 985) 5′-GTCACTCGGATCCTCAGGCTCTTTCGCCGGCATC-3′
[0670] The resulting PCR product was digested with the endonucleases NdeI and BamHI and ligated into the expression vector pET16b to create a vector for heterologous expression of BHAR.
Heterologous Expression and Purification of Recombinant Proteins
Enzymes of the BHAP
[0671] For heterologous overexpression of the AsGAT, BHAD, BHAA and ISRed enzymes, respectively, the corresponding plasmid encoding the respective enzyme was first transformed into chemically competent E. coli BL21 Al cells. The cells were then grown on LB agar plates containing 100 μg mL.sup.−1 ampicillin at 37° C. overnight. A starter culture in selective LB medium was inoculated from a single colony on the next day and left to grow overnight at 37° C. in a shaking incubator. The starter culture was used on the next day to inoculate an expression culture in selective TB medium in a 1:100 dilution. The expression culture was grown at 37° C. in a shaking incubator to an OD600 of 0.5 to 0.7, induced with 0.5 mM IPTG and 0.2% L-arabinose and subsequently grown overnight at 18° C. in a shaking incubator.
[0672] Cells were harvested at 6,000×g for 15 min at 4° C. and cell pellets were stored at −20° C. until purification of enzymes. Cell pellets were resuspended in twice their volume of buffer A (300 mM NaCl, 25 mM Tris-HCl pH 8.0, 15 mM imidazole, 1 mM β-mercaptoethanol, 0.1 mM MgCl.sub.2, 0.01 mM pyridoxalphosphate (PLP), and one tablet of SIGMAFAST™ protease inhibitor cocktail, EDTA-free per L). The cell suspension was treated with a Sonopuls GM200 sonicator (BANDELIN Electronic GmbH & Co. KG, Berlin, Germany) at an amplitude of 50% in order to lyse the cells and subsequently centrifuged at 50,000×g and 4° C. for 1 h. The filtered supernatant (0.45 μm filter, Sarstedt, Numbrecht, Germany) was loaded onto Protino® Ni-NTA Agarose (Macherey-Nagel, Düren, Germany) in a gravity column, which had previously been equilibrated with 5 column volumes of buffer A. The column was washed with 20 column volumes of buffer A and 5 column volumes of 85% buffer A and 15% buffer B and the His-tagged protein was eluted with buffer B (buffer A with 500 mM imidazole). The eluate was desalted using PD-10 desalting columns (GE Healthcare, Chicago, USA) and buffer C (100 mM NaCl, 25 mM Tris-HCl pH 8.0, 1 mM MgCl.sub.2, 0.01 mM PLP, 0.1 mM dithiothreitol (DTT)). This was followed by purification on a size exclusion column (Superdex™ 200 μg, HiLoad™ 16/600; GE Healthcare, Chicago, USA) connected to an ÄKTA Pure system (GE Healthcare, Chicago, USA) using buffer C. 2 mL concentrated protein solution was injected, and flow was kept constant at 1 mL min.sup.−1. Elution fractions containing pure protein were determined via SDS-PAGE analysis (Laemmli 1970) on 12.5% gels. Purified enzymes in buffer C were used for crystallization or stored at −20° C. in buffer C containing 50% glycerol for later use in enzymatic assays. BhcR was expressed and purified in the same way, except that buffer A contained 100 mM KCl, 20 mM HEPES-KOH pH 7.5, 10 mM MgCl.sub.2, 4 mM β-mercaptoethanol, 5% glycerol and one tablet of SIGMAFAST™ protease inhibitor cocktail, EDTA-free per L. Buffer C contained 100 mM KCl, 20 mM HEPES-KOH pH 7.5, 10 mM MgCl.sub.2, 5% glycerol and 1 mM DTT. NADH-dependent malate dehydrogenase (Mdh) and NADPH-dependent glyoxylate reductase (GhrA) from E. coli were overexpressed using the respective strains from the ASKA collection (Kitagawa, Ara et al. 2005). A starter culture in selective LB medium (34 μg mL.sup.−1 chloramphenicol) was inoculated from a single colony and left to grow overnight at 37° C. in a shaking incubator. The starter culture was used on the next day to inoculate an expression culture in selective TB medium in a 1:100 dilution. The expression culture was grown at 37° C. in a shaking incubator to an OD600 of 0.6, induced with 0.5 mM IPTG and grown for four more hours at 37° C. in a shaking incubator. The enzymes were affinity-purified in the same way as described above, except that buffer A contained 200 mM NaCl, 50 mM potassium phosphate pH 7.0, 15 mM imidazole, 1 mM β-mercaptoethanol, and one tablet of SIGMAFAST™ protease inhibitor cocktail, EDTA-free per L. Buffer C contained 100 mM NaCl, 50 mM potassium phosphate pH 7.0, and 0.1 mM DTT. The purified enzyme was stored at −20° C. in buffer C containing 50% glycerol.
[0673] Construction of T-DNA vectors for BHAP enzyme expression and localization studies The construction of all T-DNA vectors used in this study relies on the Golden Gate cloning based MoClo kit (Weber et al., PLoS ONE 2011). The MoClo kit contains a set of 95 empty standardized genetic modules that can be used for hierarchical assembly based on the Golden Gate cloning technique. This kit is comprised of plasmids for cloning: promoters, 5′ untranslated regions, signal peptides, coding sequences, and terminators. The MoClo kit can be used to assemble any eukaryotic multigene construct. In-vitro synthesized BHAP genes were matured for the Golden Gate system by removing internal BpiI and BsaI restriction enzyme sites. Golden Gate assembly was performed in a 15 μl reaction (10 U BpiI/BsaI, 400 U T4 DNA Ligase, 1×T4 DNA Ligase buffer) with a 50 cycles digestion-ligation cycle (37° C. for 2 minutes and 16° C. for 5 minutes) followed by 5 minutes at 37° C. and 10 minutes at 65° C.
[0674] The plant-codon optimized gene for the iminosuccinate reductase enzyme from Paracoccus denitrificans DSM 413 (ISRed; nucleic acid sequence shown in SEQ ID NO: 900; amino acid sequence shown in SEQ ID NO: 901) was cloned into the Level 0 vector plCH41308 by golden gate assembly. To this end, the ISRed gene was amplified with the primers
TABLE-US-00008 (SEQ ID NO: 927) 5′-ttgaagacaaaATGTACCCTTACGATGTGCCTG-3′ (SEQ ID NO: 928) 5′-ttgaagacaaaagcTCAGAGCTTAGAGATCTCGACCTC-3′
[0675] The plant-codon optimized gene for the β-hydroxyaspartate aldolase enzyme from Paracoccus denitrificans DSM 413 (BHAA; nucleic acid sequence shown in SEQ ID NO: 902; amino acid sequence shown in SEQ ID NO: 903) was cloned into the Level 0 vector pICH41308 12 by golden gate assembly. To this end, the BHAA gene was amplified with the primers
TABLE-US-00009 (SEQ ID NO: 929) 5′-ttgaagacaaaATGCATCACCATCACCACCACCGATTAGCTGTTCTCT CAGGTCATTTAAACGCTAAGACCGACTTTTC-3′ (SEQ ID NO: 930) 5′-ttgaagacaacgaaccGATCTCGACCTCTTGTGCAACAC-3′
[0676] In addition, BHAA was cloned in the Level 0 vector pAGM1299 without stop codon for C-terminal fluorescent protein fusion. To this end, the BHAA gene was amplified with the primers
TABLE-US-00010 (SEQ ID NO: 931) 5′-ttgaagacaaaATGCATCACCATCACCACCACCGATTAGCTGTTCTCT CAGGTCATTTAAACGCTAAGACCGACTTTTC-3′ (SEQ ID NO: 932) 5′-ttgaagacaacgaaccCTCGACGCAGCCAAGGAAG-3′
[0677] The plant-codon optimized gene for the β-hydroxyaspartate dehydratase enzyme from Paracoccus denitrificans DSM 413 (BHAD; nucleic acid sequence shown in SEQ ID NO: 904; amino acid sequence shown in SEQ ID NO: 905) was cloned into the Level 0 vector pICH41308 by golden gate assembly. To this end, the BHAD gene was amplified with the primers
TABLE-US-00011 (SEQ ID NO: 933) 5′-ttgaagacaaaATGCATCACCATCACCAC-3′ (SEQ ID NO: 934) 5′-ttgaagacaaaagcTCAAAGCTTGCTGTTCCAC-3′
[0678] The plant-codon optimized gene for the aspartate:glyoxylate aminotransferase enzyme from Paracoccus denitrificans DSM 413 (AsGAT; nucleic acid sequence shown in SEQ ID NO: 906; amino acid sequence shown in SEQ ID NO: 907) was cloned into the Level 0 vector pICH41308 by golden gate assembly. To this end, the AsGAT gene was amplified with the primers
TABLE-US-00012 (SEQ ID NO: 935) 5′-ttgaagacaaaATGTACCCTTACGATGTGC-3′ (SEQ ID NO: 936) 5′-ttgaagacaaaagcTCAAAGCTTAGAAGCAGCC-3′
[0679] Construction of higher-level T-DNA constructs (Level 1 & 2, Level P & M) followed the MoClo kit-based guidelines (Weber et al., 2011). The generated constructs are described in Tables 2, 3 and 4. Used promoter (p), fluorescent proteins and terminators (t) are all part of the MoClo Plant Parts kit. Used vector backbones and linker are also part of the MoClo toolkit. In addition, Arabidopsis UBIQUITIN10 promoter (UBQ10p) was added as part.
[0680] For targeting BHAP enzymes to the chloroplast and mitochondria a similar cloning strategy will be used to generate the corresponding construct for verification of the localization and implementing the pathway in plants. Regarding the localization only C-terminal fluorescent fusions can be generated due to the N-terminal localization of the target peptide for mitochondrial or chloroplastic localization.
TABLE-US-00013 TABLE 2 Constructs used for localization studies Vector/ Plant Bacterial Name Insert 1 backbone Resistance Resistance pMR211 AtUBQ10p::mCherry-ASGAT::S/RbcS3Ct pICH86966 Kanamycin Kanamycin pMR213 AtUBQ10p::mCherry-BHAD::S/RbcS3Ct pICH86966 Kanamycin Kanamycin pMR214G AtUBQ10p::eGFP-ISR::S/RbcS3Ct pICH86966 Kanamycin Kanamycin pMR261 AtUBQ10p::BHAA-mCherry::S/RbcS3Ct pICH86966 Kanamycin Kanamycin
TABLE-US-00014 TABLE 3 Constructs for in vitro enzyme assays Vector/ Bacterial Name Transcriptional unit 1 Transcriptional unit 2 Linker backbone Resistance pMR225N AtRbcS2Bp::BHAA::AtuOCSt AtRbcS1Bp::BHAD::AtuNOSt pICH50881 pAGM8031 Spectinomycin pMR226 AtRbcS3Bp::ISR::35St AtCaBp::ASGAT::S/RbcS3Ct pICH50900 pAGM8055 Spectinomycin pMR268 AtRbcS1Bp::BHAD::AtuNosT AtRbcS3Bp::ISR::35St pICH50892 pAGM8043 Spectinomycin
TABLE-US-00015 TABLE 4 Construct for stable P-hydroxyaspartate pathway implementation Name pMR228N Transcriptional unit 1 AtRbcS2Bp::BHAA::AtuOCSt Transcriptional unit 2 AtRbcS1Bp::BHAD::AtuNOSt Transcriptional unit 3 AtRbcS3Bp::ISR::35St Transcriptional unit 4 AtCaBp::ASGAT::S/RbcS3Ct Linker pICH79290 Vector/backbone pICH75322 Plant Resistance Kanamycin Bacterial Resistance Kanamycin
Transformation of Agrobacterium tumefaciens
[0681] Agrobacterium tumefaciens GV3103::pMP90 (A. tumefaciens) was transformed with corresponding T-DNA constructs by electroporation. Electrocompetent A. tumefaciens cells were thawed on ice and 1 μg plasmid DNA was added and afterwards cells were transferred into a 2 mm electroporation cuvette (Eppendorf, Germany) and electroporated using an Eppendorf electroporator 2510 (Eppendorf, Germany) set to 25 μF, 2.4 kV, 200 Ohm, and 5 msec puls length. After electroporation cells were resuspended in 1 ml YEP medium and incubated at 28° C. with shaking at 200 ppm before plating on YEP plates containing the appropriate antibiotics.
Transformation of Arabidopsis thaliana
[0682] Arabidopsis was transformed by floral dipping, which is a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. 300 ml A. tumefaciens overnight culture was pelleted at 1,600×g for 10 min at RT. The bacterial pellet was resuspended in 400 ml 5% (w/v) sucrose and supplemented with 0.02% (v/v) Silwet L-77. Arabidopsis were dipped into the A. tumefaciens suspension for 1 min and kept in the dark for 1 day before being transferred back to the growth chamber until seed harvesting.
Transient Expression of BHAP Enzymes in Nicotiana benthamiana, Protoplast Isolation and Confocal Microscopy
[0683] A. tumefaciens cultures were diluted to an OD600 of 0.4 in infiltration medium (10 mM MgCl2, 10 mM MES pH 5.7, 100 μM acetosyringon) and diluted equally for coexpression of two T-DNAs. Leaves of four-week-old greenhouse grown N. benthamiana plants were infiltrated using a syringe without a needle for transient expression. For leaf disc assays two leaf discs (1 cm diameter each) per leaf, were infiltrated similarly and harvested 5 days post infection (5 dpi).
[0684] Leaf discs were grinded, using glassbeads and windmill, and resuspended in 400 μl extraction buffer (50 mM potassium phosphate pH 7.5, 5 mM MgCl2, 1 mM EDTA, 0.1% (v/v) Triton-X 100). After centrifugation for 10 minutes at 12,000 rpm at 4° C. the supernatant was used for SDS-PAGE analysis following immunoblot analysis. For localization studies leaves were harvested two days post infection and protoplasts were isolated for confocal microscopy. Dissected leaf material was vacuum infiltrated with protoplast digestion solution (1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4 M Mannitol, 20 mM KCl, 20 mM MES pH 5.6, 10 mM CaCl2, 0.1% BSA) and incubated at 30° C. for two hours. Protoplasts were transferred to a new tube and allowed to sediment and afterwards resuspended in W5 solution (154 mM NaCl, 25 mM CaCl2, 5 mM KCl, 2 mM MES pH 5.6). Protoplasts were analysed using a Zeiss LSM 780 confocal microscope and Zeiss ZEN software. Excitation/emission wavelength settings were used as follows: CFP (458 nm/458-514 nm), GFP (488 nm/490-550 nm), mCherry (561 nm/580-625 nm), and chlorophyll A (488 nm/640-710 nm). Fiji software was used for image processing.
[0685] BHAP enzyme activity assays Each BHAP enzyme was assayed individually after 5 days transient expression in N. benthamiana. Leaf discs were grinded, using glass beads and windmill, and resuspended in 700 μl extraction buffer (50 mM potassium phosphate pH 7.5, 5 mM MgCl.sub.2, 1 mM EDTA, 0.1% (v/v) Triton-X 100). After centrifugation for 10 minutes at 12,000 rpm at 4° C. and 25 μl of the supernatant was used per enzyme assay. All assays were carried out at 30° C. in a total volume of 300 μl. The oxidation of NADH was followed at 340 nm on a Cary 60 UV-Vis photospectrometer (Agilent) in quartz cuvettes with a path length of 10 mm (Hellma Analytics).
[0686] The reaction mixture to assay AsGAT activity contained 100 mM potassium phosphate buffer pH 7.5, 0.1 mM PLP, 0.2 mM NADH, 5 mM glyoxylate, 20 mM aspartate, 25 μl of N. benthamiana leaf disc extract and 8.75 μg MDH enzyme.
[0687] The reaction mixture to assay BHAA activity contained 100 mM potassium phosphate buffer pH 7.5, 0.1 mM PLP, 0.2 mM NADH, 0.5 mM MgCl.sub.2, 5 mM glyoxylate, 10 mM glycine, 25 μl of N. benthamiana leaf disc extract and 7 μg purified BHAD and 7 μg purified ISRed enzyme.
[0688] The reaction mixture to assay BHAD activity contained 100 mM potassium phosphate buffer pH 7.5, 0.1 mM PLP, 0.2 mM NADH, 2 mM β-hydroxyaspartate (BHA), 25 μl of N. benthamiana leaf disc extract and 7 μg purified ISRed enzyme. BHA (=(2R,3S)-β-hydroxyaspartate) was custom-synthesized for the inventors by the company NewChem (Newcastle upon Tyne, United Kingdom), and was determined to be >95% pure by NMR analysis.
[0689] The reaction mixture to assay ISRed activity contained 100 mM potassium phosphate buffer pH 7.5, 0.1 mM PLP, 0.2 mM NADH, 2 mM BHA, 25 μl of N. benthamiana leaf disc extract and 7 μg purified BHAD enzyme. The formation of aspartate by ISRed activity was confirmed by LC-MS/MS. To take samples for LC-MS/MS analysis, the reaction volume of the assay was increased to 600 μl and contained 100 mM potassium phosphate buffer pH 7.5, 0.1 mM PLP, 0.2 mM NADH, 2 mM BHA, 25 μl of N. benthamiana leaf disc extract and 7 μg purified BHAD enzyme. 90 μL aliquots were taken after 0, 1, 2 and 3 minutes and the reaction was immediately stopped by addition of formic acid (4% final concentration). The samples were centrifuged at 17,000×g and 4° C. for 15 min and the supernatant was subsequently used for LC-MS analysis.
[0690] The LC-MS measurements were done using an Agilent 6550 Funnel Q-TOF LC-MS system equipped with an electrospray ionization source set to negative ionization mode. Liquid chromatography (LC) was carried out as follows: The analytes were separated on an aminopropyl column (30 mm×2 mm, particle size 3 μm, 100 Å, Luna NH.sub.2, Phenomenex inc.) using a mobile phase system comprised of 95:5 20 mM ammonium acetate pH 9.3 (adjusted with ammonium hydroxide to a final concentration of approximately 10 mM)/acetonitrile (A) and acetonitrile (B). Chromatographic separation was carried out using the following gradient condition at a flow rate of 250 μl min-1: 0 min 85% B; 3.5 min 0% B, 7 min, 0% B, 7.5 min 85% B, 8 min 85% B. Column oven and autosampler temperature were maintained at 15° C. The ESI source was set to the following parameters: Capillary voltage was set at 3.5 kV and nitrogen gas was used as nebulizing (20 psig), drying (13 l/min, 225 C) and sheath gas (12 l/min, 400° C.). The QTOF mass detector was calibrated prior to measurement using an ESI-L Low Concentration Tuning Mix (Agilent) with residuals and corrected residuals less than 2 ppm and 1 ppm respectively. MS data were acquired with a scan range of 50-600 m/z. Autorecalibration was carried out using 113 m/z as reference mass. Subsequent peak integration of all analytes was performed using the eMZed software, which is specific for the development of LCMS data analysis.
[0691] The same procedure was used to assay the activity of the enzymes purified from E. Coli BL21 implemented with the BHAP:
[0692] The above described assay conditions are used to measure individually BHAP enzyme activity in the generated Arabidopsis thaliana BHAP plants. Therefore, leaves of four-week old transgenic lines expressing the BHAP are harvested and total leaf protein is extracted as stated above. The optimal volume of crude leaf extract in the enzymatic assays needs to be optimized. In order to distinguish between aspartate formation by ISRed activity and leaf extract aspartate aminotransferase activity, .sup.15N-glycine will be used in BHAA assays as substrate to measure .sup.15N-aspartate enrichment over time by LC/MS.
Verification of BHAP Implementation in Arabidopsis thaliana.
[0693] Seeds of primary transformants with the nucleic acid construct were harvested four weeks after transformation and plated on 0.5× Murashige & Skoog (MS) medium containing kanamycin as selection marker (50 μg/ml). After stratification at 4° C. for 2 days in the dark, seeds were grown in a growth chamber (100 μmol photons m.sup.−2s.sup.−1) at high CO.sub.2 concentrations (3000 ppm) under normal day conditions (12 h day/12 h night, 22° C.). Seedlings were grown on plates for 12 days and transferred to soil and grown under the same conditions as described.
[0694] Leaves of four-week-old plants were harvested in liquid nitrogen for genomic DNA isolation. Leaf material was grinded using glass beads and windmill and resuspended in 400 μl extraction buffer (250 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS) and 150 μl 3 M potassium acetate. After vortexing samples were centrifuged at 12,000 rpm for 5 minutes. Supernatant was transferred to a new tube containing 550 μl 100% isopropanol and inverted several times. Samples were again centrifuges for 5 minutes at 12,000 rpm and the supernatant was discarded. DNA pellet was washed with 300 μl 70% (v/v) ethanol and centrifuged for 5 minutes at 12,000 rpm. The supernatant was discarded and the pellet after 10 minutes ethanol evaporation resuspended in 50 μl TE-Buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA). 2 μl of resuspended DNA was used for PCR. The complete integration of all four BHAP enzymes was verified using a combination of forward and reverse primers specific for the promoter and the coding sequence.
[0695] Integration of AsGAT under control of the chlorophyll A binding protein promoter was verified with the primers
TABLE-US-00016 (SEQ ID NO: 937) 5′-GACTAGCCAATAGCAACCTC-3′ (SEQ ID NO: 938) 5′-CTCTGATAGCAGACACGGAAT-3′
[0696] Integration of BHAA under control of the RubisCO small subunit 2B promoter was verified with the primers
TABLE-US-00017 (SEQ ID NO: 939) 5′-CCAGTAGCCATACACATTCAC-3′ (SEQ ID NO: 940) 5′-GCTTGTCGTTCACCTTGAG-3′
[0697] Integration of BHAD under control of the RubisCO small subunit 1B promoter was verified with the primers
TABLE-US-00018 (SEQ ID NO: 941) 5′-GAGCCAAAGCAACCGATC-3′ (SEQ ID NO: 942) 5′-GATCTGTAAGCGTCATCAGC-3′
[0698] Integration of ISRed under control of RubisCO small subunit 3B promoter was verified with the primers
TABLE-US-00019 (SEQ ID NO: 943) 5′-GAAAGGAGCCAAAAGCAAC-3′ (SEQ ID NO: 944) 5′-GTCCAACACCAGTTCCATC-3′
[0699] Successful gDNA isolation was confirmed by amplification of the Arabidopsis housekeeping gene ACTIN 2 (AT3G18780) using primers
TABLE-US-00020 (SEQ ID NO: 945) 5′-GTTGGGATGAACCAGAAGGA-3′ (SEQ ID NO: 946) 5′-GAACCACCGATCCAGACACT-3′
[0700] To verify homozygosity of the ggt1 mutant a gene-specific/T-DNA specific primer pair was used. Wildtype GGT1 was amplified using primers
TABLE-US-00021 (SEQ ID NO: 947) 5′-CCTTGCCCTTGGCTCTAGAACC-3′ (SEQ ID NO: 948) 5′-GTCATACCTAAACCGCCTGAAGTC-3′
[0701] The T-DNA integration in the GGT1 locus in the ggt1 mutant was verified using primers
TABLE-US-00022 (SEQ ID NO: 949) 5′-TAACTCTCCCCACTCTTTGCC-3′ (SEQ ID NO: 950) 5′-ATATTGACCATCATACTCATTGC-3′
TABLE-US-00023 TABLE 5 Description of the Sequence List SEQ ID No Description Type 1-299 IsRed enzyme Amino Acid 300-599 AsGAT enzyme Amino Acid 600-899 β-hydroxyaspartate regulatory protein Amino Acid 900 Arabidopsis codon-optimized sequence of IsRed enzyme from Nucleic acid Paracoccus denitrificans DSM 413 targeted to plant peroxisomes 901 IsRed enzyme from Paracoccus denitrificans DSM 413 containing Amino acid a plant peroxisome targeting sequence 902 Arabidopsis codon-optimized sequence of BHAA enzyme from Nucleic acid Paracoccus denitrificans DSM 413 containing a plant peroxisome targeting sequence 903 BHAA enzyme from Paracoccus denitrificans DSM 413 containing Amino acid a plant peroxisome targeting sequence 904 Arabidopsis codon-optimized sequence of BHAD enzyme from Nucleic acid Paracoccus denitrificans DSM 413 targeted to plant peroxisomes 905 BHAD enzyme from Paracoccus denitrificans DSM 413 containing Amino acid a plant peroxisome targeting sequence 906 Arabidopsis codon-optimized sequence of AsGAT enzyme from Nucleic acid Paracoccus denitrificans DSM 413 targeted to plant peroxisomes 907 AsGAT enzyme from Paracoccus denitrificans DSM containing a Amino acid plant peroxisome targeting sequence 908 Arabidopsis codon-optimized sequence of IsRed enzyme from Nucleic acid Paracoccus denitrificans DSM 413 and for cytosolic expression 909 IsRed enzyme from Paracoccus denitrificans DSM 413 for Amino acid expression in plant cytosolic expression 910 Arabidopsis codon-optimized sequence of BHAA enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 911 BHAA enzyme from Paracoccus denitrificans DSM 413 for Amino acid expression in plant cytosol 912 Arabidopsis codon-optimized sequence of BHAD enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 913 BHAD enzyme from Paracoccus denitrificans DSM 413 for Amino acid expression in plant cytosol 914 Arabidopsis codon-optimized sequence of AsGAT enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 915 AsGAT enzyme from Paracoccus denitrificans DSM 413 for Amino acid expression in plant cytosol 916 Arabidopsis Ferredoxin-2 chloroplastic target peptide for N- Nucleic acid terminal fusion to target BHAP proteins to plant chloroplasts. 917 Arabidopsis Ferredoxin-2 chloroplastic target peptide for N- Amino acid terminal fusion to target BHAP proteins to plant chloroplasts. 918 Arabidopsis serine hydroxymethyl transferase target peptide for N- Nucleic acid terminal fusion to target BHAP proteins to plant mitochondria. 919 Arabidopsis serine hydroxymethyl transferase target peptide for N- Amino acid terminal fusion to target BHAP proteins to plant mitochondria 920 Nucleic acid sequence coding Dicosoma sp. red fluorescent protein Nucleic acid (m Cherry) 921 Dicosoma sp. red fluorescent protein (mCherry) Amino acid 922 Nucleic acid sequence coding enhanced green fluorescent protein Nucleic acid 923 enhanced green fluorescent protein Amino acid 924 Agrobacterium tumefaciens NOS promoter Nucleic acid 925 Agrobacterium tumefaciens MAS promoter Nucleic acid 926 cauliflower mosaic virus 35S promoter Nucleic acid 927 primer for cloning of IsRed in pICH41308, fwd Nucleic Acid 928 primer for cloning of IsRed in pICH41308, rev Nucleic Acid 929 primer for cloning of BHAA in pICH41308, fwd Nucleic Acid 930 primer for cloning of BHAA in pICH41308, rev Nucleic Acid 931 primer for cloning of BHAA in pAGM1299 without stop codon, fwd Nucleic Acid 932 primer for cloning of BHAA in pAGM1299 without stop codon, rev Nucleic Acid 933 primer for cloning of BHAD in pICH41308, fwd Nucleic Acid 934 primer for cloning of BHAD in pICH41308, rev Nucleic Acid 935 primer for cloning of AsGAT in pICH41308, fwd Nucleic Acid 936 primer for cloning of AsGAT in pICH41308, rev Nucleic Acid 937 Primer used to assess the integration of AsGAT under control of Nucleic Acid chlorophyll A binding protein promoter, fwd 938 Primer used to assess the integration of AsGAT under control of Nucleic Acid chlorophyll A binding protein promoter, rev 939 Primer used to assess the integration of BHAA under control of Nucleic Acid RubisCO small subunit 2B promoter, fwd 940 Primer used to assess the integration of BHAA under control of Nucleic Acid RubisCO small subunit 2B promoter, rev 941 Primer used to assess the integration of BHAD under control of Nucleic Acid RubisCO small subunit 1B promoter, fwd 942 Primer used to assess the integration of BHAD under control of Nucleic Acid RubisCO small subunit 1B promoter, rev 943 Primer used to assess the integration of ISRed under control of Nucleic Acid RubisCO small subunit 3B promoter, fwd 944 Primer used to assess the integration of ISRed under control of Nucleic Acid RubisCO small subunit 3B promoter, rev 945 Primer for Actin 2 amplification, fwd Nucleic Acid 946 Primer for Actin 2 amplification, rev Nucleic Acid 947 Primer for wildtype GGT1 amplification, fwd Nucleic Acid 948 Primer for wildtype GGT1 amplification, rev Nucleic Acid 949 primer for verification of T-DNA integration in the GGT1 locus, fwd Nucleic Acid 950 primer for verification of T-DNA integration in the GGT1 locus, rev Nucleic Acid 951 nucleic acid sequence coding the peroxisomal targeting signal 2 Nucleic acid (PTS2) from Arabidopsis peroxisomal citrate synthase 3 (At2g42790) 952 peroxisomal targeting signal 2 (PTS2) from Arabidopsis Amino acid peroxisomal citrate synthase 3 (At2g42790) 953 Pden_3919 BHAA Amino acid 954 Pden_3920 BHAD Amino acid 955 BHAA enzyme derived from Paracoccus denitrificans DSM 413 Amino acid containing a plant peroxisome targeting sequence and without tag for immunoblot analysis 956 BHAD enzyme from Paracoccus denitrificans DSM 413 containing Amino acid a plant peroxisome targeting sequence and without tags for immunoblot 957 ISRed enzyme derived from Paracoccus denitrificans DSM 413 Amino acid containing a plant peroxisome targeting sequence and without tags for immunoblot 958 AsGAT enzyme derived from Paracoccus denitrificans DSM 413 Amino acid containing a plant peroxisome targeting sequence and without tags for immunoblot analysis 959 Arabidopsis codon-optimized sequence of BHAA enzyme from Nucleic acid Paracoccus denitrificans DSM 413 containing a plant peroxisome targeting sequence, and without tags for immunoblot analysis 960 Arabidopsis codon-optimized sequence of BHAD enzyme from Nucleic acid Paracoccus denitrificans DSM 413 containing a plant peroxisome targeting sequence and without tags for imunoblot analysis 961 Arabidopsis codon-optimized sequence of ISRed enzyme from Nucleic acid Paracoccus denitrificans DSM containing a plant peroxisome targeting sequence and without tags for immunoblot analysis 962 Arabidopsis codon-optimized sequence of AsGAT enzyme from Nucleic acid Paracoccus denitrificans DSM 413 containing a plant peroxisome targeting sequence, and without tags for immunoblot analysis 963 Arabidopsis codon-optimized sequence of BHAA enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 964 Arabidopsis codon-optimized sequence of BHAD enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 965 Arabidopsis codon-optimized sequence of IsRed enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 966 Arabidopsis codon-optimized sequence of AsGAT enzyme from Nucleic acid Paracoccus denitrificans DSM 413 for expression in plant cytosol 967 Conserved amino acid sequence of the IsRed enzymes Amino acid 968 Conserved amino acid sequence of the AsGAT enzymes Amino acid 969 IsRed enzyme from Paracoccus denitrificans DSM 413 Nucleic acid 970 BHAA enzyme from Paracoccus denitrificans DSM 413 Nucleic acid 971 BHAA enzyme from Paracoccus denitrificans DSM 413 Amino acid 972 BHAD enzyme from Paracoccus denitrificans DSM 413 Nucleic acid 973 BHAD enzyme from Paracoccus denitrificans DSM 413 Amino acid 974 AsGAT enzyme from Paracoccus denitrificans DSM 413 Nucleic acid 975 BHA-regulatory protein from Paracoccus denitrificans DSM 413 Nucleic acid 976 Primer for cloning of ISRed from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, fwd 977 Primer for cloning of ISRed from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, rev 978 Primer for cloning of BHAA from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, fwd 979 Primer for cloning of BHAA from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, rev 980 Primer for cloning of BHAD from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, fwd 981 Primer for cloning of BHAD from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, rev 982 Primer for cloning of AsGAT from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, fwd 983 Primer for cloning of AsGAT from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, rev 984 Primer for cloning of BHAR from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, fwd 985 Primer for cloning of BHAR from Paracoccus denitrificans DSM Nucleic acid 413 into pET16b, rev 986 multigene T-DNA construct pMR228N for BHAP implementation Nucleic acid in plant peroxisomes.
Phenotypic Analysis
[0702] Arabidopsis thaliana seeds are plated on 0.5× Murashige & Skoog (MS) medium containing kanamycin as selection marker (50 μg/ml) for segregation analysis to confirm homozygous integration of the nucleic acid construct (T-DNA). Seeds of homozygous plants are plated on 0.5× Murashige & Skoog (MS) medium without selection marker. After stratification at 4° C. for 2 days in the dark, seeds are grown in a growth chamber (100 μmol photons m.sup.−2s.sup.−1) at ambient 002 concentrations (400 ppm) under normal day conditions (12 h day/12 h night, 22° C.). Alternatively, plants are shifted between ambient CO.sub.2 concentrations (400 ppm) and high CO.sub.2 concentrations (3000 ppm) under normal day conditions (12 h day/12 h night, 22° C.). In order to determine phenotypic parameters, the plants are imaged and rosette size and leaf size areanalysed using Fiji software. Leaf fresh weight is measured by weighting leaves from comparable developmental stages.
[0703] As a matter of photorespiratory stress protection chlorophyll fluorescence (Fv/Fm) upon shift between low (100 ppm CO.sub.2), ambient and high CO.sub.2 concentrations are measured using an Imaging PAM (Walz, Germany). Seedlings are dark exposed for 15 minutes and exposed to saturating light pulse (10 000 μmol photons m.sup.−2 s.sup.−1 for 800 ms). Photosynthetic parameters are recorded with pre-illuminates plants grown at 100 μmol photons m.sup.−2 s.sup.−1 with stepwise increasing light intensities up to 1600 μmol photons m.sup.−2 s.sup.−1 using the Dual-PAM. ETR I, ETR II, NPQ, Y(NPQ), Y(NO) are calculated by the Dual-PAM 100 software.
Gas Exchange Measurements
[0704] Photosynthetic rates are measured using a LI-6400XT portable photosynthesis analyzer (LI-COR Environmental). Conditions were maintained at 1500 μmol m.sup.−2 s.sup.−1 photon flux and A/C.sub.i curves are determined by stepwise changes in external CO.sub.2 supply ranging from 50 to 2000 ppm. Based on the initial slope of the A/C.sub.i curve the maximum rate of carboxylation (V.sub.cmax) and maximum electron transport rate (J.sub.max) is calculated. Inhibition of photosynthesis by oxygen is assessed by determining the CO.sub.2 compensation point in the presence of different oxygen concentrations (2-40%). The CO.sub.2 compensation point is less affected in the BHAP implemented lines as compared to the wild type.
Transformation of Nicotiana tabacum.
[0705] The implementation of the BHAP in tobacco is achieved by transforming Nicotiana tabacum leaf discs with pMR228N as described in Gallois et al. in Methods in Molecular Biology 1995, vol. 49, p. 39.
Free Ammonium Quantification in Plant Tissue
[0706] Free ammonium was quantified in plant tissue using a colorimetric assay as previously described. In detail, 10 mg of grinded material was resuspended in 100 μL chloroform and 200 μl 100 mM HCl. Samples were rotated for 15 minutes at 4° C. and centrifuged at 12,000×g for 10 minutes at 4° C. 150 μl of the aqueous phase was transferred to a new tube containing 10 mg washed charcoal and centrifuged for 5 minutes at 4° C. at 16,000×g. 50 μl of the supernatant was mixed with 50 μl 100 mM HCl and used for further processing. Assay conditions were as follow. 20 μl of sample were mixed with 100 μl solution A, containing 1% (w/v) phenol and 0.005% (w/v) sodium nitroprusside solution in water and 100 μl solution B, containing 1% (v/v) sodium hypochlorite and 0.5% (w/v) sodium hydroxide solution in water. The assay mixture was incubated at 37° C. for 30 minutes and absorbance was measured using a Synergy™ HTX Multi-Mode Microplate Reader (BioTek). Total ammonium quantification was calculated based on a standard curve.
Metabolite Profiling
[0707] For metabolite profiling green tissue of 14 days old seedlings was harvested by immediate quenching with liquid nitrogen at the middle of the light phase. Frozen material was grinded using a precooled mortar and pestil. Grinded material was aliquoted under continuous liquid nitrogen expose to avoid sample thawing. 50 mg of leaf material is used for metabolite extraction using a one-phase extraction protocol as previously described. In detail, 1.5 ml of extraction mix, containing water:methanol:chloroform at ratio 1:2.5:1 and 5 μM ribitol as internal standard, was added to frozen material. Samples were vortexed for 20 seconds, rotated for 6 minutes at 4° C. and centrifuged for 2 minutes at 20,000×g at room temperature. The supernatant was transferred to a new tube and stored at −80° C. before further processing. For metabolite profiling by gas chromatography-mass spectrometry (GC-MS), 50 μl of extract was dried using a speed vacuum concentrator. Dried samples were placed in the Gerstel MPS 2 XL autosampler for automatic sample derivatization using methoxyamine hydrochloride and N-Methyl-N-(trimethylsilyl) trifluoroacetamide before injection. The GC-MS device is a 7200 accurate mass Q-TOF GC/MS (Agilent). For relative quantification metabolite peak areas are normalized to the internal extraction standard and the material fresh weight.
Example 1—Kinetic Characterization of the Enzymes of the β-Hydroxyaspartate Pathway (BHAP) and Reconstruction of the BHAP In Vitro
[0708] The genes encoding for the four enzymes of the BHAP in the genome of Paracoccus denitrificans DSM 413 were identified and these four proteins were heterologously expressed in E. coli. The four enzymes were purified and subjected to kinetic characterization by conducting suitable enzyme assays. Kinetic parameters of the enzymes are summarized in Table 1. The complete reaction sequence of the BHAP, catalyzed by these four enzymes, is shown in
[0709] To assess the efficiency of the BHAP in converting its input molecule (glyoxylate) into its output molecule (oxaloacetate), a combined assay of all four enzymes was conducted. Additionally, the enzymes malate dehydrogenase (MDH) and formate dehydrogenase (FDH) were added. MDH converts oxaloacetate into malate, which can be analyzed well via LC-MS, while FDH as cofactor regeneration system is required for the regeneration of the cofactor NADH by oxidation of formate to carbon dioxide. The results of this assay are shown in
[0710] Furthermore, this assay demonstrates that the concentration of the required co-substrate aspartate remains largely the same over the course of the assay. With these in vitro results the stability and effectiveness of the enzyme-catalyzed reaction network is demonstrated that is the BHAP, which suggests that the pathway can also be used with high efficiency in a host microorganism, especially since it would not deplete the intracellular aspartate pool too much.
TABLE-US-00024 TABLE 6 Kinetic data of the four enzymes of the β-hydroxyaspartate pathway. Enzyme Substrate app. k.sub.cat [s.sup.−1] app. K.sub.M [mM] app. k.sub.cat/K.sub.M [M.sup.−1 s.sup.−1] ISRed Iminosuccinate 201.04 ± 10.20 0.088 ± 0.010 2.29 * 106 NADH — 0.023 ± 0.003 — NADPH — 0.33 ± 0.05 — BHAA Glyoxylate 85.96 ± 3.64 0.23 ± 0.03 3.72 * 105 Glycine 90.98 ± 02.41 4.31 ± 0.34 2.11 * 104 (2R, 3S)-β-hydroxy- 33.11 ± 1.29 0.28 ± 0.03 1.18 * 105 aspartate D-Threonine 76.21 ± 2.49 9.24 ± 0.86 8.25 * 103 BHAD (2R, 3S)-β-hydroxy- 35.01 ± 0.82 0.20 ± 0.02 1.75 * 105 aspartate AsGAT Glyoxylate 58.07 ± 0.82 0.43 ± 0.02 1.34 * 105 L-Aspartate 56.36 ± 0.73 2.51 ± 0.10 2.25 * 104 Glycine 0.76 ± 0.01 9.52 ± 0.40 7.97 * 101 Oxaloacetate 0.76 ± 0.02 2.90 ± 0.27 2.62 * 102 L-Serine 8.82 ± 0.31 2.10 ± 0.24 4.20 * 103 L-Glutamate 5.03 ± 0.26 20.62 ± 2.33 2.44 * 102
Example 2—Designing the β-Hydroxyaspartate Pathway (BHAP) for Implementation in Plant Peroxisomes
[0711] Initially, the β-hydroxyaspartate pathway (BHAP) from Paracoccus denitrificans DSM 413 has been implemented in plant peroxisomes due to high expected concentrations of photorespiration-derived glyoxylate (
TABLE-US-00025 TABLE 7 Required enzymes to convert 2-phosphoglycolate into oxaloacetate in the projected photorespiratory bypass. Substrates and products of all enzymes are given, with possible cofactors indicated in brackets. Reaction/ EC no. of Examples Natural Enzyme enzyme to be Enzyme to for plant no. employed be employed organisms localization Substrate(s) Product(s) Reference a 2.6.1.X aspartate-glyoxylate Paracoccus — aspartate, glyoxylate oxaloacetate, (not yet given) transaminase denitrificans glycine b 4.1.3.14 erythro-β- Paracoccus — glyoxylate, glycine erythro-β- hydroxyaspartate aldolase denitrificans hydroxyaspartate c 4.3.1.20 erythro-β- Paracoccus — erythro-β- iminosuccinate, H.sub.2O Biochem. J. (former 4.2.1.38) hydroxyaspartate denitrificans hydroxyaspartate 1965, 97(2), dehydratase 547 d 1.5.1.X iminosuccinate reductase Paracoccus — iminosuccinate aspartate (NAD) (not yet given) denitrificans (NADH)
Example 3—Demonstrating BHAP Enzyme Targeting to Plant Peroxisomes
[0712] The inventors were able to demonstrate the correct localization of BHAP enzymes in plant peroxisomes (
Example 4—BHAP Enzymatic Activity in Plants
[0713] The inventors were able to demonstrate activity of each of the BHAP enzymes in N. benthamiana leaf extracts (
[0714] BHAP enzymes were individually tested for enzymatic activity in plant cell extracts of plant lines with transient expression of each enzyme, and compared to WT plant cell extracts. BHAP enzymes were transiently expressed in N. benthamiana leaf discs under the same photosynthetic promoter as contained in the multigene T-DNA construct for stable implementation of the BHAP in Arabidopsis (pMR228N,
Example 5—Multigene BHAP T-DNA Construct for in Planta Implementation and Verification of Construct Functionality
[0715] The inventors constructed a multigene T-DNA nucleic acid construct for implementation of the BHAP in plants (
Example 6—BHAP Implementation in Plants
[0716] Arabidopsis wildtype Col-0 was transformed with the generated BHAP T-DNA construct by floral dipping as described in the Methods. In order to facilitate high pathway flux via the BHAP, the synthetic pathway was also implemented in the Arabidopsis ggt1 mutant (GK-649H07). The ggt1 mutant is deficient in the peroxisomal glyoxylate:glutamate aminotransferase 1 (GGT1, At1g23310) and accumulates glyoxylate. The inventors verified complete BHAP implementation in Arabidopsis WT and ggt1 mutant by genotyping using for each BHAP enzyme a combination of forward and reverse primers specific for the promoter and the coding sequence, as described in the method section (
[0717] These data strongly suggest that the BHAP can successfully be implemented also in other C3 plant, as oil, cereal, food or biomass crop (Table 8).
TABLE-US-00026 TABLE 8 Overview of potential plant species used for implementation of the BHAP. Notably, the crop species presented here are all C3 photosynthesis type crops where photorespiration plays a major role in limiting plant productivity. Scientific name Common name Purpose Helianthus annuus Sunflower Oil crop Brassica napus Rapeseed/canola Oil crop Camelina sativa Camelina Oil crop Oryza sativa Rice Cereal crop Hordeum vulgare Barley Cereal crop Triticum spp. Wheat Cereal crop Avena sativa Oat Cereal crop Solanum lycopersicum Tomato Food crop Solanum tuberosum Potato Food crop Ipomoea batatas Sweet Potatoes Food crop Glycine max Soybean Food crop Beta vulgaris Sugar beet Food crop Psidium guajava Common guava Food crop Citrus limon Lemon Food crop Mangifera indica Mango Food crop Allium cepa Onion Food crop Pisum sativum Pea Food crop Secale cereale Roggen Food crop Canavalia ensiformis Jack bean Food crop Medicago sativa L. Alfaalfa Food crop Prunus amygdalus L. Almond Food crop Phaseolus vulgaris L. Bean Food crop Malus spp. Apple Food crop Prunus armeniaca L. Apricot Food crop Asparagus officinalis L. Asparagus Food crop Persea american P.mill. Avocado Food crop Musa sapientum L. Banana Food crop Brassica oleracea L. Cabbage Food crop Daucus carota L. Carrot Food crop Anacardium occidentale L. Cashew Food crop Cicer arietinum L. Chickpea Food crop Theobroma cacao L. Cocoa Food crop Vigna unguiculata Cowpea Food crop Vaccinium macro carpon Cranberry Food crop Cucumis sativus L. Cucumber Food crop Solanum melongena L. Eggplant Food crop Vicia faba L. Faba bean Food crop Ficus carica L. Fig Food crop Linum usitatissimum L. Flaxseed Food crop Vitis vinifera L. Grape Food crop Lactuca spp. lettuce Food crop Phaseolus lunatus L. Lima bean Food crop Beta vulgaris L. Mangold Food crop Olea europea L. Olive Food crop Citrus sinensis L. Orange Food crop Petroselium crispum Parsley Food crop Prunus persica L. Peach Food crop Arachis hypogea L. Peanut Food crop Pyrus communis L. Pear Food crop Carya illinoinensis Pecan Food crop Capsicum spp. Pepper Food crop Cajanus cajan Pigeonpea Food crop Prunus spp. Plum Food crop Gossypium hirsutum L. Cotton Fiber crop Ocimum tenuiflorum Tulsi Medicinal crop Ricinus communis L. Castor bean Medicinal crop Taraxacum officinale Dandelion Medicinal crop Nicotiana tabacum Tobacco Biomass crop Arabidopsis thaliana Thale cress Scientific research
Example 7—Establishing Transgenic BHAP Plant Lines
[0718] The inventors verified BHAP implementation in Arabidopsis thaliana WT Col-0 (
Example 8—Phenotypic Analysis of BHAP Plants
[0719] The physiological consequences of the BHAP, as CO.sub.2 neutral photorespiratory bypass, were tested by analyzing the phenotype of the established BHAP Arabidopsis thaliana plants. This included the quantification of phenotypic parameters like plant growth, rosette size and leaf size, as well as the measurement of free ammonium in green tissue. In all cases, the transgenic plants engineered with the BHAP performed markedly better than the ggt1 mutant (background), which was used as negative control (
Example 9—Metabolomic Analysis of BHAP Plants
[0720] Metabolic flux through the BHAP was tested by quantifying cellular aspartate levels, as BHAP intermediate, and malate levels as described in the Method section. As expected, aspartate and malate levels were significantly increased in plant lines engineered with the BHAP under photorespiratory conditions (
Example 10—Gas Exchange Measurements in BHAP Plants
[0721] In order to confirm the improved carbon efficiency of BHAP plants photorespiratorily released CO.sub.2 will be measured by gas exchange and quantified as described by (Long, S P and Bernacchi, CJ Journal of Experimental Botany 2003). A lowered photorespiratory CO.sub.2 loss will be observed in BHAP plants. Furthermore, respiratory CO.sub.2 release will be determined to exclude enhanced respiratory CO.sub.2 release in BHAP plants. The improved carbon efficiency of the BHAP plants results in a lowered CO.sub.2 compensation point.
Example 11—BHAP Implementation in Plants Cytoplasm, Chloroplast or Mitochondria
[0722] The implementation of BHAP in cellular compartments such as mitochondria, chloroplast and cytosol is possible and of interest.
[0723] The implementation of BHAP in plant cell cytosol can be accomplished by targeting BHAP enzymes to the cytosol using the nucleotide sequences coding the polypeptides with the enzymatic activities (a)-(d) without a specific organellar targeting sequences (IsRed: SEQ ID NOs 908 and 909, BHAA: SEQ ID NOs 910 and 911, BHAD: SEQ ID NOs 912 and 913, and AsGAT: SEQ ID NOs 914 and 915).
[0724] As glyoxylate is low in chloroplast and mitochondria, the establishing of the BHAP requires the expression of glycolate dehydrogenase (EC 1.1.99.14) (enzymatic activity (e)) for glyoxylate production, fused to chloroplast or mitochondria targeting sequences.
[0725] Thus, establishing the BHAP in mitochondria requires only one additional gene for functionality, since produced oxaloacetate is directly metabolized by the TCA cycle.
[0726] BHAP expression in chloroplasts requires further an ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) (enzymatic activity (f)) for downstream metabolism of oxaloacetate. Oxaloacetate produced by the BHAP will be decarboxylated by PEPCK and generated phosphoenolpyruvate will be converted into 2-phosphoglycerate by plastidial enolase, ENO1 (At1g74030 (http://doi.wiley.com/10.1016/j.febslet.2009.02.017)) and further into 3-phosphoglycerate by plastidial phosphoglycerate mutase (PGM, At5g51820). Produced 3-phosphoglycerate subsequently enters photosynthetic carbon metabolism. Although the decarboxylation of oxaloacetate in the chloroplast might positively influence CO.sub.2 assimilation, the PEPCK step requires one ATP per produced phosphoenolpyruvate, thereby weakening the energy-conserving principle of the BHAP. Furthermore, produced PEP might be distributed between 3-phosphoglycerate production and the shikimate pathway for aromatic amino acid biosynthesis. Alternatively, oxaloacetate is reduced by a NAD(P)-dependent malate dehydrogenase to produce malate that can be exported from the chloroplast via Dit1 (At5g12860).
[0727] Therefore, the implementation of BHAP in plant cell mitochondria can be accomplished by targeting BHAP enzymes to the mitochondria using the nucleotide sequences coding the polypeptides with the enzymatic activities (a) erythro-β-hydroxyaspartate aldolase, (b) erythro-β-hydroxyaspartate dehydratase, (c) iminosuccinate reductase, (d) aspartate-glyoxylate transaminase, and (e) glycolate dehydrogenase fused to a N-terminal targeting sequence such as the serine hydroxymethyltransferase 1 (At4g37930) (SEQ ID No 919).
[0728] The implementation of BHAP in plant cell chloroplast can be accomplished by targeting BHAP enzymes to the chloroplast using the nucleotide sequences coding the polypeptides with the enzymatic activities (a) erythro-β-hydroxyaspartate aldolase, (b) erythro-β-hydroxyaspartate dehydratase, (c) iminosuccinate reductase, (d) aspartate-glyoxylate transaminase, (e) glycolate dehydrogenase, and (f) phosphoenolpyruvate carboxykinase fused to a N-terminal targeting sequence such as the Arabidopsis ferredoxin-2 (At1 g60950) (SEQ ID No 917).
Example 12—Implementing the BHAP as a Heterologous Photorespiration Bypass Pathway into Cyanobacteria
[0729] The four genes of the BHAP were successfully integrated at two different neutral sites of the S. elongatus PCC7942 chromosome. This was done both in the WT strain and in a deletion strain that lacks the genes necessary for the formation of carboxysomes (ccmK-O), and therefore requires elevated atmospheric CO.sub.2 concentrations for growth (this strain is henceforth referred to as ΔK-O). Subsequently, successful expression of the pathway enzymes was verified by measuring enzyme activities in cell-free extracts.
[0730] As shown in
[0731] Next, it was tested whether the implementation of the BHAP in the ΔK-O background conferred a specific phenotype due to improved capabilities for photorespiration. To this end, three replicate cultures (50 mL in baffled shake flasks) of each strain were grown at 30° C. in a light incubator in an atmosphere containing 0.5% CO.sub.2, and growth curves were recorded. This experiment was independently repeated three times;
[0732] For all experiments, the slope of the growth curves during the linear growth phase was determined. As shown in Table 9, implementation of the BHAP in the ΔK-O background resulted in significantly increased slopes compared to the control strain in all cases. Taken together, these results confirm that implementation of the BHAP in the ΔK-O background permits faster growth of the engineered strain.
TABLE-US-00027 TABLE 9 Slopes derived from cyanobacterial growth curves. Suitable intervals of the growth curves were fitted with linear regression. The average slope ± standard deviation of three replicates is given, and it was compared whether the slopes of the two strains were significantly different in each experiment. Interval for linear Slope ΔK-O + Significant Experiment regression [h] Slope ΔK-O BHAP difference? 30° C., I 80-190 0.080 ± 0.002 0.096 ± 0.003 Yes; p = 0.0004 30° C., II 45-140 0.070 ± 0.002 0.086 ± 0.001 Yes; p < 0.0001 30° C., III 45-125 0.076 ± 0.002 0.092 ± 0.002 Yes; p = 0.0002 37° C. 90-190 0.062 ± 0.002 0.074 ± 0.001 Yes; p < 0.0001
Example 13—Alternative Approaches for BHAP Implementation in Plants
[0733] An alternative to the detailed expression of the BHAP under photosynthetic promoters is the constitutive active expression using the Agrobacterium NOS (SEQ ID No: 924) and MAS promoter (SEQ ID No: 925), the cauliflower mosaic virus 35S promoter (SEQ ID No: 926) and the Arabidopsis UBIQUITIN10 promoter. This approach allows BHAP expression in photosynthetic and heterotrophic tissue as well as a time/light independent expression. It is worth mentioning that the implementation of non-native plant promoters increases potential effects of post-transcriptional gene silencing.
Example 14—Implementation of BHAP in Nicotiana tabacum
[0734] The enzymes of the BHAP are successfully expressed in Nicotiana tabacum using the above-described plant transformation methods. Enzyme activity of each BHAP enzyme is maintained in Nicotiana tabacum.
Example 15—Characterization of BHAC Enzymes from Other Bacterial Species
[0735] 5 enzymes each are chosen from the provided list of iminosuccinate reductase sequences (SEQ ID 1-299) and aspartate-glyoxylate aminotransferase sequences (SEQ ID 300-599). These enzymes are cloned, expressed, and purified according to the protocols described in the Methods section. Subsequent enzymatic assays demonstrate that these BHAC enzymes from other bacterial species indeed have the respective activity of iminosuccinate reductases or aspartate-glyoxylate aminotransferases, as suggested. Their kinetic parameters are largely in accordance with the previously measured parameters of the enzymes from Paracoccus denitrificans. These results underline that the provided list of sequences indeed contains a multitude of enzymes with the proposed activities, which could be used in various combinations to engineer a functional BHAC into plant hosts.