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WO2025153518A1 - Moyens et procédés de production de plantes ligneuses à composition de lignine modifiée - Google Patents

Moyens et procédés de production de plantes ligneuses à composition de lignine modifiée

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Publication number
WO2025153518A1
WO2025153518A1 PCT/EP2025/050854 EP2025050854W WO2025153518A1 WO 2025153518 A1 WO2025153518 A1 WO 2025153518A1 EP 2025050854 W EP2025050854 W EP 2025050854W WO 2025153518 A1 WO2025153518 A1 WO 2025153518A1
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Prior art keywords
plant
cistronic
plants
seq
expression construct
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Wout Boerjan
Nette DE RIDDER
Lennart HOENGENAERT
Ruben VANHOLME
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01208—4-Hydroxycoumarin synthase (2.3.1.208)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Non-edible lignocellulosic biomass is the most abundant renewable carbon source on earth, and therefore a promising candidate. It consists primarily of carbohydrates (cellulose and hemicellulose) and an aromatic fraction (lignin).
  • lignin an aromatic fraction
  • current lignocellulosic feedstock suffers from a relatively low processing efficiency, mainly because of the presence of the recalcitrant lignin polymer, even after pretreatment steps (Chen and Dixon, 2007; Yuan et al., 2021).
  • FIG. 5 Endpoint measurement of saccharification assay on SCOP1, SCOP2 and SCOP3 transgenic poplars. Alkali pretreated wood powder was incubated with a cellulase mixture for 72 hours.
  • C Two independent SCOP2 lines (batch2) displayed a significant increase in sugar release compared to WT.
  • D SCOP3 line (batch 2) shows no significant difference in sugar release compared to WT. Significant differences are indicated by * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001 (Welch's t-test).
  • CWR cell wall residue.
  • the invention provides a multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme, a nucleotide sequence encoding a 2A peptide, a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme and a plant terminator sequence wherein the coumarin synthase enzyme in the multi-cistronic chimeric expression construct lacks its natural stop codon sequence.
  • the invention provides a multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme followed by a nucleotide sequence encoding a 2A peptide followed by a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme followed by a plant terminator sequence and wherein the coumarin synthase enzyme in the multi-cistronic chimeric expression construct lacks its natural stop codon sequence.
  • the invention provides a multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme, a spacer sequence, a nucleotide sequence encoding a 2A peptide, a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme and a plant terminator sequence wherein the coumarin synthase enzyme in the multi-cistronic chimeric expression construct lacks its natural stop codon sequence.
  • the coumarin synthase enzyme is depicted in SEQ ID NO: 2.
  • the spacer sequence in the multi-cistronic chimeric expression construct is a nucleotide sequence encoding for GSG.
  • the spacer sequence in the multi-cistronic chimeric expression construct is a nucleotide sequence depicted in SEQ ID NO: 24.
  • the plant expressible promoter present in the multi-cistronic chimeric expression construct is a plant promoter active in cells that make a secondary cell wall.
  • the plant expressible promoter present in the multi-cistronic chimeric expression construct is the cellulose synthase 8-B promoter.
  • cellulose synthase 8-B promoter is depicted in SEQ ID NO: 1.
  • the feruloyl-CoA-6'-hydroxylase enzyme is the feruloyl-CoA-6'-hydroxylase- 1 enzyme or the feruloyl-CoA-6'-hydroxylase-2 enzyme.
  • a hardwood selected from the group consisting of acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple, sycamore, ginkgo, palm trees, and sweet gum; or
  • agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743).
  • Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.
  • Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • plants used as a model like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • the transformation of plants by means of Agro bacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White
  • the transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229], Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and nontransformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion). Examples l.lntroduction of the scopoletin biosynthetic pathway in poplar
  • constructs differ by the order of the F6'H1 and COSY genes.
  • the order of F6'H and COSY genes in the SCOP2 construct is the same as in the construct successfully used by Hoengenaert et al (2022) in Arabidopsis.
  • the third construct pCesA8B:F6'Hl (SCOP3) aimed at expressing F6'H alone. Because the reaction catalyzed by COSY is also catalyzed by light, we wanted to investigate with this latter construct whether solely F6'H is sufficient to boost scopoletin biosynthesis in poplar wood.
  • the three SCOP constructs were transferred into poplar via Agrobacterium-m ediated transformation.
  • a total of 52 independent transgenic lines were generated: twenty-one lines with SCOP1, seventeen with SCOP2 and fourteen lines containing SCOP3.
  • poplars containing the SCOP3 construct exhibited a severely dwarfed phenotype ultimately resulting in the death of twelve out of the fourteen independent lines.
  • transgenic poplars were analyzed in two separate batches.
  • batch 1 one plant per independent line was transferred from in vitro to soil to perform preliminary experiments to select the most interesting lines that would be further analyzed with the proper number of repeats per independent line.
  • batch 2 a selection of poplars was included based on findings from the first batch and planted in soil with five replicates per independent line. Additionally, four and five independent lines, that were not included in batch 1, were introduced in batch 2 for SCOP1 and SCOP3, respectively, with five repeats per independent line.
  • SCOP1 transgenic poplars contained more scopoletin and scopolin (scopoletin glucoside) ( Figure 3) than SCOP2 and SCOP3 transgenic poplars, highlighting that the position of the genes relative to the T2A sequence as well as the presence of COSY play a crucial role in the production of scopoletin.
  • SCOP2 and SCOP3 lines produce products that are toxic to the cells.
  • levels of the glucoside of 6-hydroxyferulic acid the latter being the de-esterification product of 6- hydroxyferuloyl CoA, is higher in SCOP2 transgenic poplars compared to WT and SCOP1 and SCOP3 poplars (see Figure 3).
  • SCOP3-1 failed to produce 6-hydroxyferulic acid, scopoletin, or scopolin, therefore the presence of the SCOP3 construct was analyzed using PCR.
  • SCOP3-1 The one surviving line with the SCOP3 construct, SCOP3-1, was included in the selection of batch 1, even though no scopoletin and only marginal levels of scopolin were detected, this was the only line of this construct that survived in this batch. 4.Lignin analysis of transgenic poplar trees
  • SCOP 1 lines produce significantly most scopoletin and scopolin, and less 6-hydroxyferulic acid hexose, while normal growth is maintained in most lines.
  • SCOP2 lines produce less scopoletin and scopolin than SCOP1 lines, and accumulate more ferulic acid hexose, derived from the intermediate 6- hydroxy feruloyl-CoA.
  • the SCOP2 lines that had improved saccharification were all dwarfed, whereas all but one SCOP1 line that had improved saccharification had normal growth. Plants that had normal growth and improved saccharification were only obtained from the SCOP1 construct where COSY is positioned upstream of F6'H.
  • SCOP1 lines (SCOP1-2, SCOP1-7, and SCOP1-8) previously demonstrated promising results in greenhouse settings, including a respective increase of 29, 22 and 15% in saccharification efficiency compared to wild-type (WT) control. Therefore, a field trial was started on May 29, 2024, in Wetteren, Belgium, where we aimed to evaluate their performance under outdoor conditions. The field trial, designed with a randomized complete block design, comprised five blocks with eight replicates per genotype per block, resulting in a total of 160 poplars). Border plants were included to minimize edge effects but were not included in the experimental analyses.
  • SEQ ID NO: 4 Capsella rubella: Carub.0001s2711 (92.3% identity with SEQ ID NO: 2)
  • SEQ ID NO: 6 Durio zibethinus: Duzib235G0533 (63.5% identity with SEQ ID NO: 2)
  • SEQ ID NO: 7 Populus trichocarpa: Potri.004G053500 (62.9% identity with SEQ ID NO: 2)
  • SEQ ID NO: 8 Eucalyptus grandis: Eucgr.D00550 (55.0% identity with SEQ ID NO: 2)
  • SEQ ID NO: 9 Arabidopsis thaliana
  • SEQ ID NO: 10 Arabidopsis lyrate: AL3G25540 (96.4% identity with SEQ ID NO: 9)
  • SEQ ID NO: 11 Capsella rubella: Carub.0003sl307 (93.4% identity with SEQ ID NO: 9) MAPTLSTAQFSTPAEVTDFVVHRGNGVKGLSETGIKALPDQYIQPLEERLINKFVNETDEAIPVIDMSNPDEKKVAEAVC
  • SEQ ID NO: 12 Arabidopsis thaliana: AT1G55290 (77.4% identity with SEQ ID NO: 9)
  • the wild types reached heights of approximately 200 cm after 23 weeks of growth in soil.
  • all poplars were harvested by cutting the stem 12 cm above the soil level leaving 2-3 axillary buds to allow development of new shoots.
  • the bottom 10 cm of the harvested stem were debarked, the wood cut in pieces of about 1 cm, frozen and stored in -70°C for phenolic profiling.
  • the leftover stem piece was weighed, debarked, air-dried, weighed, and ground in a ball mill for cell wall analysis and saccharification.
  • Soluble phenolic compounds were extracted from approximately 15 mg of stem material with 1 mL methanol at room temperature for 15 min under 750 rpm shaking. After centrifugation at room temperature and maximum speed 800 pL of supernatant was transferred in a new Eppendorf and dried at reduced pressure in a SpeedVac and subsequently solubilized in 100 pL of cyclohexane and 100 pL Milli-Q. water. Samples were subjected to Ultra Performance Liquid Chromatography High Resolution Mass Spectrometry (UPLC-HRMS) at the VIB Metabolomics Core Ghent (VIB-MCG).
  • UPLC-HRMS Ultra Performance Liquid Chromatography High Resolution Mass Spectrometry
  • the flow rate was set to 0.35 mL/min.
  • Electrospray Ionization (ESI) was applied, LockSpray ion source was operated in negative ionization mode under the following specific conditions: capillary voltage, 2.5 kV; reference capillary voltage, 2.5 kV; source temperature, 120°C; desolvation gas temperature, 550°C; desolvation gas flow, 800 L/h; and cone gas flow, 50 L/h.
  • the collision energy for full MS scan was set at 6 eV for low energy settings, for high energy settings (HDMSe) it was ramped from 20 to 70 eV. For DDA-MSMS the low mass ramp was ramped between 15-35 eV, and the high mass ramp was ramped between 35-70 eV.
  • Mass range was set from 50 to 1500 Da in full MS with a scan time of 0.1 s and from 100 to 1500 Da in DDA-MSMS with a scan time of 0.2 s.
  • Nitrogen greater than 99.5%
  • Leucine-enkephalin 100 pg/pL solubilized in watenacetonitrile 1:1 [v/v], with 0.1% formic acid
  • Profile data was recorded through Unifi Workstation v2.0 (Waters). Data processing was done with Progenesis QI v3.0 (Waters). Data processing was performed with Progenesis QI software version 3.0 (Waters) for chromatogram alignment and compound ion detection. The detection limit was set at maximum sensitivity.
  • CWR The preparation of CWR involved a series of sequential extraction steps.
  • ground wood powder was sequentially washed for 30 min each with Milli-Q water at 98°C, ethanol at 76°C, chloroform at 59°C and acetone at 54°C.
  • the remaining CWR was dried under vacuum and was determined gravimetrically (expressed as mass percentage of dry weight).
  • the lignin content was determined using the gravimetry-based Klason assay. Klason lignin was determined on 50 mg of ground wood powder per sample and performed according to National Renewable Energy Laboratory (Sluiter et al., 2012). Briefly, 0.5 mL of 72% sulphuric acid was added to 50 mg of CWR in glass vials and stirred with a glass rod while incubating at 30°C for 1 hour. Next, 14 mL water was added to reach a final concentration of 4% sulphuric acid. These samples were subsequently transferred to 50 mL bottles and are autoclaved for lh at 121°C. After autoclaving, these samples were stored at 4°C for 16 hours.
  • the filter papers were transferred to a muffle furnace following a specific temperature program (12 min at 105°C, 10°C/min to 205°C, 30 min at 250°C, 20°C/min to 575°C, 180 min at 575°C, cool down at room temperature in a desiccator for 1 hour) and weighed to calculate the ash-corrected lignin content.
  • the acid-soluble lignin was determined using a spectrophotometer (Genesys 10 S UV-VIS, Thermo-Scientific) by measuring absorbance at 205 nm.
  • the acid-soluble lignin content was calculated with the Lambert-Beer law (Dence, 1992). Saccharification assays
  • the saccharification assay was performed according to (Van Acker et al., 2016). In short, 10 mg of wood powder was weighed in individual Eppendorf using an analytical balance (XPE105; Mettler-Toledo). Different biomass pretreatments were performed, alkaline pretreatment (62.5 mM NaOH) and acid pretreatment (1 M HCI). Afterwards, the biomass (pretreated as well as not pretreated) was washed by adding 1 mL of 70% EtOH which was incubated for 17 hours at 55°C. Next, the pellet was washed another three times by each time adding 1 mL 70% EtOH and washed a last time with 1 mL acetone. The pellet was afterwards dried in a SpeedVac.
  • the actual saccharification starts by adding 1 mL of acetic acid buffer (pH 4.7) and 100 pL of 100 times diluted cellulase enzyme mixture (Cellic CTect2, Sigma) to the dried biomass at 50°C.
  • the activity of the enzyme mixture was determined using a filter paper assay, and 0.005 and 0.007 filter paper units were added to each sample in the first and second saccharification assay, respectively.
  • Glucose release was determined at five different time points (2h, 6h, 24h, 48h and 72h after enzyme addition) using GOD-POD where glucose was determined using a spectrophotometer by measuring absorbance at 405 nm after which the concentration was calculated using the Lambert-Beer law.

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Abstract

La présente invention concerne des gènes chimériques et des constructions qui peuvent être utilisés pour modifier la composition de lignine de plantes ligneuses et augmenter la saccharification et l'efficacité de dépulpage desdites plantes. En particulier, la composition de lignine est modifiée par incorporation de scopolétine.
PCT/EP2025/050854 2024-01-16 2025-01-15 Moyens et procédés de production de plantes ligneuses à composition de lignine modifiée Pending WO2025153518A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071670A2 (fr) * 1999-05-21 2000-11-30 Board Of Control Of Michigan Technological University Procede accroissant la cellulose et modifiant la biosynthese de la lignine dans les plantes
EP1198985A1 (fr) 1999-07-22 2002-04-24 Japan as represented by Dir. Gen. of National Inst. of Agrobiological Resources,Ministry of Agriculture, Forestry and Fisherie Procede de transformation ultrarapide de monocotyledon
WO2018046526A1 (fr) * 2016-09-06 2018-03-15 Vib Vzw Moyens et procédés pour augmenter la production de coumarine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071670A2 (fr) * 1999-05-21 2000-11-30 Board Of Control Of Michigan Technological University Procede accroissant la cellulose et modifiant la biosynthese de la lignine dans les plantes
EP1198985A1 (fr) 1999-07-22 2002-04-24 Japan as represented by Dir. Gen. of National Inst. of Agrobiological Resources,Ministry of Agriculture, Forestry and Fisherie Procede de transformation ultrarapide de monocotyledon
WO2018046526A1 (fr) * 2016-09-06 2018-03-15 Vib Vzw Moyens et procédés pour augmenter la production de coumarine

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* Cited by examiner, † Cited by third party
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