ITRM20110588A1 - USE OF PLANTS WITH A REDUCED LEVEL OF HOMOGALACTURONANE DE-ESTERIFIED IN THE CELL WALL OR PARTS OF THEM TO IMPROVE THE SACCARIFICATION OF VEGETABLE BIOMASSES - Google Patents
USE OF PLANTS WITH A REDUCED LEVEL OF HOMOGALACTURONANE DE-ESTERIFIED IN THE CELL WALL OR PARTS OF THEM TO IMPROVE THE SACCARIFICATION OF VEGETABLE BIOMASSES Download PDFInfo
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- ITRM20110588A1 ITRM20110588A1 IT000588A ITRM20110588A ITRM20110588A1 IT RM20110588 A1 ITRM20110588 A1 IT RM20110588A1 IT 000588 A IT000588 A IT 000588A IT RM20110588 A ITRM20110588 A IT RM20110588A IT RM20110588 A1 ITRM20110588 A1 IT RM20110588A1
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Classifications
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- 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
- 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)
Description
DESCRIZIONE DESCRIPTION
a corredo di una domanda di brevetto per invenzione divisionale della domanda 6 dal titolo: accompanying a patent application for divisional invention of application 6 entitled:
“Uso di piante con un ridotto livello di omogalatturonano de-esterificato nella parete cellulare o parti di esse per migliorare la saccarificazione di biomasse vegetali” "Use of plants with a reduced level of de-esterified homogalacturonan in the cell wall or parts of them to improve the saccharification of plant biomass"
La presente invenzione concerne un procedimento per una migliore conversione in zuccheri fermentabili di biomasse lignocellulosiche, un processo chiamato saccarificazione, che permette poi la produzione, anche su scala industriale, di tutto ciò che può essere ottenuto per via fermentativa, ivi compreso l’etanolo (bioetanolo). The present invention relates to a process for a better conversion of lignocellulosic biomass into fermentable sugars, a process called saccharification, which then allows the production, even on an industrial scale, of everything that can be obtained by fermentation, including ethanol ( bioethanol).
La presente invenzione riguarda l’espressione stabile in piante di enzimi pectinolitici e di inibitori della pectina metilesterasi per aumentare la degradabilità dei tessuti vegetali tramite digestione enzimatica e quindi l’efficienza di saccarificazione. The present invention relates to the stable expression in plants of pectinolytic enzymes and inhibitors of pectin methylesterase to increase the degradability of plant tissues through enzymatic digestion and therefore the saccharification efficiency.
La crescente richiesta di carburanti alternativi ai derivati del petrolio si dirige a processi per la produzione di biocarburanti. La produzione di etanolo e biogas attraverso il processo di digestione a partire da matrici organiche complesse di natura biologica (biomasse) provenienti da filiere agricole (per es. biomasse residue di coltivazioni di interesse alimentare o industriale e rifiuti di origine agroindustriale o urbana) rappresenta un promettente modo di produrre energia rinnovabile e costituisce una delle vie preferenziali di smaltimento dei residui e rifiuti organici. Una considerevole porzione delle biomasse (circa 75%) è rappresentata dalle pareti cellulari delle piante. Queste sono costituite da una matrice eterogenea composta da carboidrati polimerici associati ad altri componenti, come lignina e proteine. I polisaccaridi di parete mediante degradazione producono zuccheri semplici (saccarificazione) che possono poi essere utilizzati dai microrganismi impiegati nelle fermentazioni aerobica e anaerobica per la produzione di bioetanolo e biogas, rispettivamente. Attualmente l’idrolisi enzimatica è considerata la tecnologia più promettente e a minore impatto ambientale per la “saccarificazione”, cioè la conversione di materiale vegetale grezzo in zuccheri fermentabili. Il fattore limitante in questo processo è dato dalla naturale resistenza delle pareti cellulari alla degradazione enzimatica. Le tecnologie di pre-trattamento attualmente disponibili per rendere le biomasse accessibili agli enzimi idrolitici che degradano i singoli componenti della parete vegetale sono costose e in alcuni casi richiedono l’utilizzo di sostanze tossiche e/o inquinanti, come acidi, perossidi e ammoniaca, spesso insieme a processi di disgregazione meccanica. The growing demand for alternative fuels to petroleum derivatives is headed for processes for the production of biofuels. The production of ethanol and biogas through the digestion process starting from complex organic matrices of biological nature (biomass) from agricultural supply chains (e.g. residual biomass of crops of food or industrial interest and waste of agro-industrial or urban origin) represents a promising way of producing renewable energy and constitutes one of the preferential ways of disposing of residues and organic waste. A considerable portion of the biomass (about 75%) is represented by the cell walls of plants. These consist of a heterogeneous matrix composed of polymeric carbohydrates associated with other components, such as lignin and proteins. Wall polysaccharides by degradation produce simple sugars (saccharification) which can then be used by microorganisms used in aerobic and anaerobic fermentation for the production of bioethanol and biogas, respectively. Currently, enzymatic hydrolysis is considered the most promising technology with the lowest environmental impact for "saccharification", that is, the conversion of raw plant material into fermentable sugars. The limiting factor in this process is the natural resistance of the cell walls to enzymatic degradation. The pre-treatment technologies currently available to make biomass accessible to hydrolytic enzymes that degrade the individual components of the plant wall are expensive and in some cases require the use of toxic and / or polluting substances, such as acids, peroxides and ammonia, often together with mechanical disintegration processes.
La disponibilità di materiale vegetale più facilmente degradabile migliorerebbe considerevolmente l’utilizzo delle biomasse e ridurrebbe la necessità di pretrattamenti costosi e a forte impatto ambientale. Poiché l’accessibilità della componente cellulosica agli enzimi degradativi è impedita dalla presenza delle pectine, delle emicellulose e della lignina, modificazioni qualitative e quantitative di questi componenti nelle pareti vegetali potrebbero migliorare l’efficienza della saccarificazione. The availability of more easily degradable plant material would considerably improve the use of biomass and reduce the need for expensive pre-treatments with a high environmental impact. Since the accessibility of the cellulosic component to degradative enzymes is prevented by the presence of pectins, hemicelluloses and lignin, qualitative and quantitative modifications of these components in the plant walls could improve the efficiency of saccharification.
Il problema tecnico che l’invenzione si propone di risolvere è costituito dalla resistenza delle pareti cellulari presenti nelle biomasse lignocellulosiche all’idrolisi enzimatica da parte di cellulasi e altri enzimi degradativi. The technical problem that the invention aims to solve is the resistance of the cell walls present in the lignocellulosic biomass to enzymatic hydrolysis by cellulase and other degradative enzymes.
Gli autori hanno messo a punto un procedimento che riduce, nella parete cellulare di una pianta, la presenza della forma de-esterificata deN’omo-galatturonano, un componente delle pectine vegetali, mediante l’espressione di una poligalatturonasi esogena, per esempio la poligalatturonasi fungina (PG), e/o la sovraespressione di un inibitore della pectina metil esterasi (PMEI), e hanno ottenuto un aumento dell’efficacia dell’idrolisi enzimatica della cellulosa. The authors have developed a procedure which reduces, in the cell wall of a plant, the presence of the de-esterified form of homo-galacturonan, a component of plant pectins, through the expression of an exogenous polygalacturonase, for example the polygalacturonase fungal (PG), and / or overexpression of a pectin methyl esterase (PMEI) inhibitor, and achieved an increase in the efficacy of enzymatic hydrolysis of cellulose.
Le biomasse contenenti la pectina così modificata non necessitano di pretrattamenti costosi e inquinanti e consentono di migliorare il procedimento di saccarificazione. The biomasses containing the modified pectin do not require expensive and polluting pretreatments and allow to improve the saccharification process.
Stato della tecnica State of the art
Le biomasse vegetali, cioè piante e/o parti di esse ad ogni stato vegetativo e essiccativo, sono una delle risorse alternative di energia, e di materiali e prodotti industriali rinnovabili. Le pareti delle cellule vegetali comprendono circa il 75% in peso di biomassa lignocellulosica (1) e possono essere una abbondante fonte potenziale di etanolo (2-4). Sono composte di una matrice eterogenea di polimeri di carboidrati associati con altri componenti, quali la lignina e alcune proteine. La degradazione della parete cellulare in zuccheri fermentabili, detta saccarificazione, costituisce il processo chiave per la produzione di etanolo da tali biomasse. L’idrolisi enzimatica è la tecnologia più promettente e compatibile con la salvaguardia deN’ambiente per la saccarificazione (5,6), soffrendo però di forti limitazioni, specialmente per una sua applicazione a livello industriale, proprio a causa della resistenza delle pareti cellulari all'idrolisi (7). Tra le cause di tale resistenza sono da elencare: l’eterogeneità e la complessità dei componenti strutturali delle pareti cellulari stesse, e le loro interazioni interpolimeriche (8); la minore efficacia degli enzimi per i substrati insolubili, componenti delle pareti (9); la presenza nelle pareti di inibitori di enzimi microbici (10); il grado di lignificazione (11). Vegetable biomass, that is plants and / or parts of them in any vegetative and drying state, are one of the alternative sources of energy, and of renewable industrial materials and products. Plant cell walls comprise about 75% by weight of lignocellulosic biomass (1) and can be an abundant potential source of ethanol (2-4). They are composed of a heterogeneous matrix of carbohydrate polymers associated with other components, such as lignin and some proteins. The degradation of the cell wall into fermentable sugars, called saccharification, is the key process for the production of ethanol from these biomasses. Enzymatic hydrolysis is the most promising technology and compatible with the protection of the environment for saccharification (5,6), but it suffers from severe limitations, especially for its application on an industrial level, precisely because of the resistance of the cell walls to hydrolysis (7). Among the causes of this resistance are to be listed: the heterogeneity and complexity of the structural components of the cell walls themselves, and their interpolymeric interactions (8); the lower efficacy of enzymes for insoluble substrates, components of the walls (9); the presence in the walls of inhibitors of microbial enzymes (10); the degree of lignification (11).
Sono al momento pertanto richiesti pretrattamenti costosi, che fanno uso di prodotti chimici tossici come acidi, perossidi o ammoniaca, spesso accompagnati da passaggi di rottura meccanica, per rendere le biomasse vegetali accessibili ad enzimi che degradano la parete cellulare (CWDEs). Un esempio di pretrattamento è rappresentato dalla idrolisi con acido solforico 1.3% (p/p) ed autoclavatura a 130*C per 40 min (14) Tali pretrattamenti sono, in alcuni casi, anche inefficienti, perché causano la degradazione di prodotti potenzialmente utili che si possono ottenere durante la degradazione delle biomasse. Therefore, expensive pre-treatments are currently required, which make use of toxic chemicals such as acids, peroxides or ammonia, often accompanied by mechanical breaking steps, to make plant biomass accessible to enzymes that degrade the cell wall (CWDEs). An example of pretreatment is represented by hydrolysis with 1.3% sulfuric acid (w / w) and autoclaving at 130 * C for 40 min (14) These pretreatments are, in some cases, even inefficient, because they cause the degradation of potentially useful products that can be obtained during the degradation of biomass.
Risulta pertanto evidente come la riduzione o eliminazione di tali pretrattamenti costituirebbe una significativa miglioria al procedimento di saccarificazione (12). A tal fine la modifica della struttura della parete cellulare costituisce una alternativa biotecnologica non inquinante (13). It is therefore evident that the reduction or elimination of these pretreatments would constitute a significant improvement to the saccharification process (12). To this end, the modification of the cell wall structure constitutes a non-polluting biotechnological alternative (13).
E’ stato dimostrato, per esempio, che la riduzione del contenuto di lignina nelle pareti cellulari di erba medica migliora il processo di saccarificazione da esse ma riduce la crescita delle piante (14). It has been shown, for example, that reducing the lignin content in the cell walls of alfalfa improves the saccharification process from them but reduces plant growth (14).
Un componente polisaccarico particolarmente critico nelle dicotiledoni per l’integrità dei tessuti e l’accessibilità a enzimi CWDEs è il reticolo di pectina coesiva che avvolge la cellulosa-emicellulosa, che a sua volta contiene i principali elementi che conferiscono resistenza. E’ anche noto che i legami intermolecolari delle pectine influenzano la plasticità delle pareti (15-17) e che la forma acida dell’omogalatturonano (HGA) forma legami crociati con gli ioni Calcio (Ca<++>) per formare “zone di giunzione” che irrigidiscono la parete cellulare (18-20). La quantità di HGA, il suo livello di metilazione e la distribuzione dei gruppi metilici influenza la formazione di tali "zone di giunzione”. HGA è sintetizzato e secreto in una forma molto metil esterificata, incapace di formare il reticolo con ioni Ca<++>(21). Successivamente viene de-esterificato da pectine metil-esterasi (PMEs), enzimi localizzati nelle pareti, che producono lunghe catene di residui carbossilici liberi, in grado di interagire con gli ioni Ca<++>, e formare così le “zone di giunzione” (22). A particularly critical polysaccharic component in dicotyledons for tissue integrity and accessibility to CWDEs enzymes is the cohesive pectin network that surrounds the cellulose-hemicellulose, which in turn contains the main elements that confer resistance. It is also known that the intermolecular bonds of pectins influence the plasticity of the walls (15-17) and that the acid form of homogalacturonan (HGA) cross-links with Calcium ions (Ca <++>) to form "zones of junction ”which stiffen the cell wall (18-20). The amount of HGA, its level of methylation and the distribution of methyl groups influence the formation of these "junction zones." HGA is synthesized and secreted in a highly methyl esterified form, unable to form the lattice with Ca <++ ions > (21). Subsequently it is de-esterified by pectin methyl-esterase (PMEs), enzymes located in the walls, which produce long chains of free carboxylic residues, able to interact with Ca <++> ions, and thus form the "Junction areas" (22).
La de-esterificazione dei galatturonani nei tessuti lignificati potrebbe anche far aumentare la formazione complesso lignina-carboidrati per formazione di legami benzil-estere i quali potrebbero causare un’ulteriore resistenza della parete cellulare (29). The de-esterification of galacturonans in lignified tissues could also increase the lignin-carbohydrate complex formation by forming benzyl-ester bonds which could cause further resistance of the cell wall (29).
Descrizione dell’invenzione Description of the invention
Gli autori della presente invenzione hanno sorprendentemente trovato che il processo di saccarificazione di biomasse vegetali può essere migliorato riducendo le regioni di HGA de-esterificate nella pectina, diminuendo la caratteristica collosa delle lamelle mediane, ricche di pectine, che uniscono le pareti cellulari di due cellule adiacenti. The authors of the present invention have surprisingly found that the saccharification process of plant biomass can be improved by reducing the de-esterified regions of HGA in the pectin, decreasing the sticky characteristic of the median lamellae, rich in pectin, which join the cell walls of two cells. adjacent.
Forma pertanto oggetto della presente invenzione l’uso di piante aventi un contenuto ridotto di HGA de-esterificato nella componente pectica delle pareti vegetali rispetto alle piante selvatiche, o parti di esse, per la produzione di biomasse vegetali da sottoporre a processi di saccarificazione. Therefore, the subject of the present invention is the use of plants having a reduced content of de-esterified HGA in the pectic component of the plant walls compared to wild plants, or parts of them, for the production of plant biomass to be subjected to saccharification processes.
Le piante a contenuto ridotto di HGA de-esterificato nelle pectine sono ottenibili con diverse modalità, tutte comprese nell’ambito di protezione dell’invenzione. Esse sono prodotte per via transgenica, trasformando le piante selvatiche con un gene codificante per un agente in grado di ridurre il contenuto di HGA deesterificato nelle pectine. Plants with a reduced content of HGA de-esterified in pectins can be obtained in different ways, all included within the scope of the invention. They are produced transgenically, transforming wild plants with a gene encoding an agent capable of reducing the content of deesterified HGA in pectins.
Come particolare forma di attuazione le piante sono trasformate con un acido nucleico di sequenza nucleotidica codificante per una poligalatturonasi, preferibilmente da Aspergillus niger, più preferibilmente una muteina (variante) di esso, tale da codificare per un enzima a ridotta attività specifica; ancora più preferibilmente una muteina (variante) della sequenza del gene pgall (GenBank ID N. XM 001397030, NT166530), preferibilmente deleta della sequenza dal nt. 1 al nt. 81 codificante per il peptide segnale di 21 aa. e per il propeptide dall’aa. 22 all’aa. 27), e con modifiche tali da avere una delezione deN’amminoacido treonina in posizione 34 e una sostituzione del residuo amminoacidico asparagina 178 con un aspartato (N178D). Tali posizioni si riferiscono alla proteina nativa non matura. La sequenza nucleotidica codificante per la proteina matura mutata ( pgallm ) è la seguente (SEQ ID No. 1): As a particular embodiment the plants are transformed with a nucleic acid of nucleotide sequence coding for a polygalacturonase, preferably from Aspergillus niger, more preferably a mutein (variant) thereof, such as to code for an enzyme with reduced specific activity; even more preferably a mutein (variant) of the pgall gene sequence (GenBank ID N. XM 001397030, NT166530), preferably deleted from the sequence from nt. 1 to nt. 81 encoding the 21 aa signal peptide. and for the propeptide from aa. 22 to the academic year 27), and with modifications such as to have a deletion of the amino acid threonine in position 34 and a replacement of the amino acid residue asparagine 178 with an aspartate (N178D). These positions refer to the native unripe protein. The nucleotide sequence encoding the mutated mature protein (pgallm) is the following (SEQ ID No. 1):
GACAGCTGCACGTTCACCGCTGCCGCTGCTAAAGCGGGCAAGGCGAAATGCTCTACTATC ACCCTTAACAACATCGAAGTTCCAGCTGGAACCACCCTCGACCTGACCGGTCTCACCAGC GGTACCAAGGTCATCTTCGAGGGCACCACGACCTTCCAGTACGAAGAATGGGCAGGCCCC TTGATCTCCATGAGTGGCGAACATATCACCGTCACTGGTGCCTCCGGCCACCTCATCAAT TGCGATGGTGCGCGCTGGTGGGATGGCAAGGGAACCAGCGGAAAGAAGAAGCCCAAGTTC TTTTACGCCCATGGCCTTGACTCCTCGTCTATTACTGGATTAAACATCAAAAACACCCCC CTTATGGCGTTTAGTGTCCAGGCGAATGACATTACGTTTACCGATGTTACCATCAATAAT GCGGATGGCGACACCCAGGGTGGACACGACACTGATGCGTTCGATGTTGGCAACTCGGTC GGGGTGAATATCATTAAGCCTTGGGTCCATAACCAGGATGACTGTCTTGCGGTTAACTCT GGCGAGAACATCTGGTTCACCGGCGGCACCTGCATTGGCGGCCACGGTCTCTCCATCGGC TCTGTCGGCGACCGCTCCAACAACGTCGTCAAGAACGTCACCATCGAACACTCCACCGTG AGCAATTCCGAAAACGCCGTCCGAATTAAGACCATCTCTGGCGCCACTGGCTCCGTGTCC GAGATTACGTACTCCAACATCGTCATGTCTGGCATCTCCGATTACGGCGTGGTCATTCAG CAGGATTACGAAGACGGCAAGCCTACGGGTAAGCCGACGAACGGTGTCACTATTCAGGAT GTTAAGCTGGAGAGCGTGACTGGTAGCGTGGATAGTGGGGCTACTGAGATCTATCTTCTT TGCGGGTCTGGTAGCTGCTCGGACTGGACCTGGGACGATGTGAAAGTTACCGGGGGGAAG AAGTCCACCGCTTGCAAGAACTTCCCTTCGGTGGCCTCTTGTTAG. GACAGCTGCACGTTCACCGCTGCCGCTGCTAAAGCGGGCAAGGCGAAATGCTCTACTATC ACCCTTAACAACATCGAAGTTCCAGCTGGAACCACCCTCGACCTGACCGGTCTCACCAGC GGTACCAAGGTCATCTTCGAGGGCACCACGACCTTCCAGTACGAAGAATGGGCAGGCCCC TTGATCTCCATGAGTGGCGAACATATCACCGTCACTGGTGCCTCCGGCCACCTCATCAAT TGCGATGGTGCGCGCTGGTGGGATGGCAAGGGAACCAGCGGAAAGAAGAAGCCCAAGTTC TTTTACGCCCATGGCCTTGACTCCTCGTCTATTACTGGATTAAACATCAAAAACACCCCC CTTATGGCGTTTAGTGTCCAGGCGAATGACATTACGTTTACCGATGTTACCATCAATAAT GCGGATGGCGACACCCAGGGTGGACACGACACTGATGCGTTCGATGTTGGCAACTCGGTC GGGGTGAATATCATTAAGCCTTGGGTCCATAACCAGGATGACTGTCTTGCGGTTAACTCT GGCGAGAACATCTGGTTCACCGGCGGCACCTGCATTGGCGGCCACGGTCTCTCCATCGGC TCTGTCGGCGACCGCTCCAACAACGTCGTCAAGAACGTCACCATCGAACACTCCACCGTG AGCAATTCCGAAAACGCCGTCCGAATTAAGACCATCTCTGGCGCCACTGGCTCCGTGTCC GAGATTACGTACTCCAACATCGTCATGTCTGGCATCTCCGATTACGGCGTGGTCATTCAG CAGGATTACGAAGACGGCAAGCCTACGGGTAAGCCGACGAACGGTGTCACTATTCAGGAT GTTAAGCTGGAGAGCGTGACTGGTAGCGTGGATAGTGGGGCTACTGAGATCTATCTTCTT TGCGGGTCTGGTAGCTGCTCGGACTGGACCTGGGACGATGTGAAAGTTACCGGGGGGAAG AAGTCCACCGCTTGCAAGAACTTC CCTTCGGTGGCCTCTTGTTAG.
La sequenza amminoacidica codificante la proteina mutata matura ( pgallm ) è la seguente (SEQ ID No. 2): The amino acid sequence encoding the mature mutated protein (pgallm) is the following (SEQ ID No. 2):
DSCTFTAAAA KAGKAKCSTI TLNNIEVPAG TTLDLTGLTS GTKVIFEGTT TFQYEEWAGP LISMSGEHIT VTGASGHLIN CDGARWWDGK GTSGKKKPKF FYAHGLDSSS ITGLNIKNTP LMAFSVQAND ITFTDVTINN ADGDTQGGHD TDAFDVGNSV GVNIIKPWVH NQDDCLAVNS GENIWFTGGT CIGGHGLSIG SVGDRSNNVV KNVTIEHSTV SNSENAVRIK TISGATGSVS EITYSNIVMS DSCTFTAAAA KAGKAKCSTI TLNNIEVPAG TTLDLTGLTS GTKVIFEGTT TFQYEEWAGP LISMSGEHIT VTGASGHLIN CDGARWWDGK GTSGKKKPKF FYAHGLDSSS ITGLNIKNTP LMAFSVQAND ITFTDVTINN ADGDTQGGHD TDAFDVGNSV GVNIIKPWVH NQDDCLAVNS GENIWFTGGT CIGGHGLSIG SVGDRSNNVV KNVTIEHSTV SNSENAVRIK TISGATGSVS EITYSNIVMS
GlSDYGVVIQ QDYEDGKPTG KPTNGVTIQD VKLESVTGSV DSGATEIYLL CGSGSCSDWT WDDVKVTGGK KSTACKNFPS VASC. GlSDYGVVIQ QDYEDGKPTG KPTNGVTIQD VKLESVTGSV DSGATEIYLL CGSGSCSDWT WDDVKVTGGK KSTACKNFPS VASC.
Come esempio alternativo le piante sono trasformate con un gene codificante un inibitore delle pectine metil esterasi, preferibilmente di origine vegetale, più preferibilmente di sequenza (Locus tag: At3g17220; NCBI n. NM 112599 e NP 188348). As an alternative example the plants are transformed with a gene encoding a pectin methyl esterase inhibitor, preferably of vegetable origin, more preferably of sequence (Locus tag: At3g17220; NCBI n. NM 112599 and NP 188348).
In alternativa le piante a contenuto ridotto di HGA de-esterificato nelle pectine sono selezionate da varianti naturali o indotte da mutagenesi. Alternatively, plants with a reduced content of HGA de-esterified in pectins are selected from natural variants or induced by mutagenesis.
E’ anche neN’ambito dell’invenzione l’uso di piante già a contenuto ridotto di HGA de-esterificato rese transgeniche come sopra descritto, o piante doppie transgeniche. Also within the scope of the invention is the use of plants already with a reduced content of de-esterified HGA made transgenic as described above, or transgenic double plants.
La presente invenzione verrà ora descritta in riferimento a esempi esplicativi ma non limitativi dell’ambito di protezione. The present invention will now be described with reference to explanatory but not limiting examples of the scope of protection.
Legende delle figure Legends of the figures
Figura 1. Saccarificazione di piante PG e PMEI. Si trattano i tessuti fogliari di piante non trasformate (WT) e transgeniche con cellulasi (Celluclast 1.5L) e si misura l’efficienza dell’idrolisi enzimatica, espressa come percentuale di zuccheri totali rilasciati nel mezzo, ai tempi indicati. (A), piante di tabacco WT e esprimenti AnPGII (PG); (B), Arabidopsis WT e esprimenti pgallm (PG); (C), Arabidopsis WT e esprimenti AtPMEI2 (PMEI); (D), saccarificazione di tessuti fogliari di piante WT, PG e PMEI pre-trattate con acidi diluiti prima dell’idrolisi enzimatica. Le barre rappresentano la media ± s.e.m (n>6). Gli asterischi indicano differenze statisticamente significative tra piante WT e transgeniche, secondo il test t di Student (<*>, P<0.05;<***>, P<0.01). I pannelli negli inserti mostrano la macerazione di campioni rappresentativi di WT e transgeniche dopo 48 ore di digestione. Figure 1. Saccharification of PG and PMEI plants. The leaf tissues of untransformed (WT) and transgenic plants are treated with cellulase (Celluclast 1.5L) and the efficiency of enzymatic hydrolysis is measured, expressed as a percentage of total sugars released in the medium, at the times indicated. (A), WT tobacco plants and AnPGII (PG) expressing; (B), Arabidopsis WT and pgallm (PG) expressing; (C), Arabidopsis WT and AtPMEI2 (PMEI) expressing; (D), saccharification of leaf tissues of WT, PG and PMEI plants pre-treated with diluted acids before enzymatic hydrolysis. The bars represent the mean ± s.e.m (n> 6). Asterisks indicate statistically significant differences between WT and transgenic plants, according to Student's t test (<*>, P <0.05; <***>, P <0.01). The panels in the inserts show the maceration of representative WT and transgenic samples after 48 hours of digestion.
Figura 2. Analisi immunodot di frazioni di parete cellulare. Frazioni di parete cellulare arricchite in pectine (ChASS Chelating Agents Soluble Solids) estratte da materiale di parete cellulare da foglie di piante WT o PMEI e AnPGII sono state applicate su nitrocellulosa alle diluizioni indicate e saggiate con anticorpi monoclonali diversi. Figure 2. Immunodot analysis of cell wall fractions. Cell wall fractions enriched in pectins (ChASS Chelating Agents Soluble Solids) extracted from cell wall material from leaves of WT or PMEI and AnPGII plants were applied on nitrocellulose at the indicated dilutions and tested with different monoclonal antibodies.
Materiali e metodi Materials and methods
Piante transgeniche. Piante di Arabidopsis thaliana, ecotipo Columbia (Col-0), sono state ottenute da G. Redei e A.R. Kranz (Arabidopsis Information Service, Frankfurt, Germania). La generazione di piante PMEI e di piante di tabacco esprimenti pgallm è stata descritta (23, 24). Per l’espressione transgenica di pgallm in Arabidopsis, è stata usata una cassetta di espressione comprendente il promotore del 35S del Cauliflower Mosaic Virus (CaMV) del vettore binario pBI121 (Stratagene; GenBank ID AF485783), il gene codificante pgallm fuso al peptide segnale di PGIP1 da Phaseolus vulgaris (corrispondente ai primi 87 nt. della sequenza X64769 (30) e il terminatore del gene nopalina sintetasi (NOS) del vettore pBI121 , escissa dal costrutto descritto in (2) per doppia digestione del plasmide con Pstl e EcoRI. Il frammento di DNA escisso è stato clonato nel vettore binario pCAMBIA3300 (CAMBIA, Canberra, Australia) e il vettore ricombinante usato per trasformare il ceppo GV3101 (pMP90RK) di Agrobacterium tumefaciens per elettroporazione (31). Piante di Arabidopsis di 4 settimane (ecotipo Col-0) sono state stabilmente trasformate con il metodo della trasformazione dei primordi fiorali (32). Le piante transgeniche T1 sono state selezionate su suolo dopo irrorazione con BASTA (300 uM fosfinitricina, PPT). Gli individui resistenti sono stati trasferiti su suolo senza erbicida e i semi raccolti. La progenie T2 è stata selezionata su terreno Murashige-Skoog solido sterile con 8 mg L<1>di PPT, e le linee che con un rapporto di segregazione di 3:1 per la resistenza a PPT sono state selezionate per analisi. Le linee omozigoti sono state analizzate per espressione proteica e attività con analisi Western blot e saggio di diffusione in agar come descritto in (24). La crescita delle piante WT e trasformate è stata effettuata in camere ad ambiente controllato, a 22*C, 70% di umidità relativa con un fotoperiodo di 16-h luce e 8-h buio (100 μιτιοΙ m<2>s<1>di luce fluorescente). Transgenic plants. Plants of Arabidopsis thaliana, Columbia ecotype (Col-0), were obtained by G. Redei and A.R. Kranz (Arabidopsis Information Service, Frankfurt, Germany). The generation of PMEI plants and pgallm-expressing tobacco plants has been described (23, 24). For the transgenic expression of pgallm in Arabidopsis, an expression cassette comprising the 35S promoter of the Cauliflower Mosaic Virus (CaMV) of the binary vector pBI121 (Stratagene; GenBank ID AF485783) was used, the gene encoding pgallm fused to the signal peptide of PGIP1 from Phaseolus vulgaris (corresponding to the first 87 nt. Of the sequence X64769 (30) and the terminator of the nopalin synthetase (NOS) gene of the pBI121 vector, excised from the construct described in (2) by double digestion of the plasmid with Pstl and EcoRI. excised DNA fragment was cloned into the binary vector pCAMBIA3300 (CAMBIA, Canberra, Australia) and the recombinant vector used to transform Agrobacterium tumefaciens strain GV3101 (pMP90RK) by electroporation (31). 0) were stably transformed by the method of transformation of the flower primordia (32). The T1 transgenic plants were selected on soil after spraying with ENOUGH (300 uM Phosphinitricin, PPT). Resistant individuals were transferred to herbicide-free soil and the seeds collected. T2 progeny was selected on sterile solid Murashige-Skoog medium with 8 mg L <1> PPT, and lines with a 3: 1 segregation ratio for resistance to PPT were selected for analysis. The homozygous lines were analyzed for protein expression and activity with Western blot analysis and agar diffusion assay as described in (24). Growth of WT and transformed plants was carried out in controlled environment chambers, at 22 * C, 70% relative humidity with a photoperiod of 16-h light and 8-h dark (100 μιτιοΙ m <2> s <1> of fluorescent light).
Per la quantificazione del peso fresco (FW) e peso secco (DW) della rosetta dopo 15 gg. di crescita, le piante sono state trasferite ad un fotoperiodo di 12-h (100 m<2>s<1>di luce fluorescente). For the quantification of the fresh weight (FW) and dry weight (DW) of the rosette after 15 days. of growth, the plants were transferred to a 12-h photoperiod (100 m <2> s <1> of fluorescent light).
Le piante di Tabacco sono state cresciute in serra a 23*C e 60% di umidità relativa con un fotoperiodo di 16-h luce e 8-h buio (130 μιτιοΙ m<2>s<1>di luce fluorescente). Tobacco plants were grown in greenhouses at 23 * C and 60% relative humidity with a photoperiod of 16-h light and 8-h dark (130 μιτιοΙ m <2> s <1> of fluorescent light).
Pretrattamento chimico. Il materiale fogliare è stato miscelato con acido solforico diluito (concentrazione finale 1.3% ) e pre-trattato a 110°C per 20 min. Dopo il pre-trattamento, gli idrolizzati sono stati separati e raccolti per filtrazione e la biomassa residua lavata con acqua. Chemical pretreatment. The leaf material was mixed with diluted sulfuric acid (final concentration 1.3%) and pre-treated at 110 ° C for 20 min. After the pre-treatment, the hydrolysates were separated and collected by filtration and the residual biomass washed with water.
Idrolisi enzimatica. Campioni fogliari (100mg di peso fresco sterilizzati in una soluzione di 1% sodio ipoclorito per 1 min e lavati due volte con acqua sterile per evitare contaminazioni microbiche) sono stati incubati per 20 ore a 37*C in una soluzione contenente 50 mM tampone sodio acetato pH 5.5, e 0.5% Celluclast 1.5 L (cellulasi da Trichoderma reeser, Sigma, St Louis, MO), già sterilizzata per filtrazione. Gli zuccheri con estremità riducenti rilasciati in soluzione sono stati quantificati con il saggio PAHBAH assay (4) dopo centrifugazione. Gli zuccheri totali prima dell'idrolisi enzimatica sono stati determinati con il metodo Dubois (33). Enzymatic hydrolysis. Leaf samples (100mg fresh weight sterilized in 1% sodium hypochlorite solution for 1 min and washed twice with sterile water to avoid microbial contamination) were incubated for 20 hours at 37 * C in a solution containing 50 mM sodium acetate buffer pH 5.5, and 0.5% Celluclast 1.5 L (cellulase from Trichoderma reeser, Sigma, St Louis, MO), already sterilized by filtration. Reducing-end sugars released in solution were quantified with the PAHBAH assay (4) after centrifugation. Total sugars before enzymatic hydrolysis were determined by the Dubois method (33).
Saggio immunologico sulle pectine. Procedura. AIS (Solidi insolubili in alcol) sono stati estratti come descritto in (23). Dopo un trattamento con cloroformio:metanolo, il materiale è stato trattato due volte 80% acetone e essiccato aN’aria. Per ottenere frazioni di solidi solubili in agenti chelanti (ChASS), gli AIS (circa 10 mg) sono stati omogeneizzati due volte in un tampone contenente 50 mM TRIS-HCI e 50 mM acido Trans-1 ,2-Cicloesanodiamminotetraacetico (CDTA) pH 7.2, a 80*0. Dopo centrifugazione a 10.000 rpm per 10 min, i due sopranatanti sono stati uniti e liofilizzati. Riquadri di 6 x 6 mm sono stati marcati su membrane di nitrocellulosa (Amersham, UK) con una matita e quantità eguali di frazioni ChASS da ciascuna linea sono state disciolte in acqua e inoculate nei riquadri disegnati sulla nitrocellulosa, rispettivamente in diluizioni di circa 3x. Epitopi peptici specifici sono stati evidenziati con gli ab monoclonali PAM1 (25, 26) e JIM5 (26) (forniti dal Prof. P.Knox University of Leeds). Pectin immunoassay. Procedure. AIS (Alcohol Insoluble Solids) were extracted as described in (23). After a treatment with chloroform: methanol, the material was treated twice 80% acetone and dried in air. To obtain fractions of soluble solids in chelating agents (ChASS), the AIS (approximately 10 mg) were homogenized twice in a buffer containing 50 mM TRIS-HCI and 50 mM Trans-1, 2-Cyclohexanodiaminetetraacetic acid (CDTA) pH 7.2 , at 80 * 0. After centrifugation at 10,000 rpm for 10 min, the two supernatants were combined and lyophilized. Boxes of 6 x 6 mm were marked on nitrocellulose membranes (Amersham, UK) with a pencil and equal amounts of ChASS fractions from each line were dissolved in water and inoculated into the boxes drawn on the nitrocellulose, respectively in dilutions of approximately 3x. Specific peptic epitopes were detected with the monoclonal ab PAM1 (25, 26) and JIM5 (26) (provided by Prof. P. Knox University of Leeds).
Le membrane sono state bloccate in MPBS (1X PBS con 3% “Membrane blocking reagent powder”, Amersham, UK) per 1 ora prima dell’incubazione con gli ab primari (supernatanti degli ibridami di JIM5, JIM7 e LM7 diluiti 1/10 o di PAM1 diluito 1/20 in 3% MPBS) per 1.5 h. Dopo lavaggio in 1X PBS, le membrane sono state incubate con l’ab secondario (anti-ratto coniugato con perossidasi da rafano, Amersham, UK) diluito 1/1000 per JIM5, e con un anticorpo anti-istidina coniugato con perossidasi da rafano (Sigma A-7058) diluito 1/1000 per PAM1. Le membrane sono state lavate come descritto e successivamente trattate con il reagente ECL (Amersham, UK) per la rilevazione dell’attività perossidasica. Membranes were blocked in MPBS (1X PBS with 3% "Membrane blocking reagent powder", Amersham, UK) for 1 hour before incubation with primary ab (supernatants of JIM5, JIM7 and LM7 hybrids diluted 1/10 or of PAM1 diluted 1/20 in 3% MPBS) for 1.5 h. After washing in 1X PBS, the membranes were incubated with secondary AB (horseradish peroxidase conjugated anti-rat, Amersham, UK) diluted 1/1000 for JIM5, and with an anti-histidine antibody conjugated with horseradish peroxidase ( Sigma A-7058) diluted 1/1000 for PAM1. The membranes were washed as described and subsequently treated with the ECL reagent (Amersham, UK) for the detection of peroxidase activity.
Risultati Results
Gli autori hanno analizzato l'efficacia della saccarificazione da tessuti fogliari di piante transgeniche esprimenti una poligalatturonasi da Aspergillus niger (piante PG) (23) e di piante sovraesprimenti un inibitore della PME (piante PMEI) (24). Le piante PG presentano livelli ridotti di HGA, mentre le piante PMEI hanno un’attività ridotta di PME ed un’aumentata mutilazione di HGA. The authors analyzed the efficacy of saccharification from leaf tissues of transgenic plants expressing a polygalacturonase from Aspergillus niger (PG plants) (23) and of plants overexpressing a PME inhibitor (PMEI plants) (24). PG plants have reduced levels of HGA, while PMEI plants have reduced PME activity and increased HGA mutilation.
Sia in piante PG di Arabidopsis o di tabacco (Fig 1A and B) e in piante PMEI di Arabidopsis (Fig. 1C), il trattamento dei tessuti fogliari con cellulasi commerciali (Celluclast 1.5L) per 24 ore dà luogo ad un rilascio di zuccheri solubili più elevato che nelle rispettive piante di controllo. Both in Arabidopsis or tobacco PG plants (Fig 1A and B) and in Arabidopsis PMEI plants (Fig.1C), treatment of leaf tissues with commercial cellulases (Celluclast 1.5L) for 24 hours results in a release of sugars solubles higher than in the respective control plants.
La saccarificazione delle piante PG e PMEI è accompagnata da un’aumentata macerazione dei tessuti (si vedano inserti nelle Fig. 1A-C). Dopo un’incubazione di 24 ore in assenza di cellulasi non si osservano né rilascio di zuccheri, né macerazione dei tessuti, a prova che la riduzione del contenuto di HGA o della sua metilazione non provoca di per sé il disassemblaggio dei tessuti e la saccarificazione, ma piuttosto la capacità delle cellulasi di degradare la cellulosa nel tessuto intatto. Inoltre l’efficacia dell’idrolisi enzimatica sulle foglie pretrattate con acidi di piante selvatiche (WT) o transgeniche (PG e PMEI) non differisce in maniera significativa (Fig. 1 D). E’ da notare che, dopo 24 ore di saccarificazione, l’efficacia di idrolisi enzimatica ottenuta con foglie pre-trattate con acidi di tutte le piante è la stessa di quella osservata con le piante transgeniche non pre-trattate. Nessun significativo rilascio di zuccheri è ottenuto dopo il solo pre-trattamento acido, a prescindere dalle piante utilizzate. I risultati indicano che la degradabilità della cellulosa è migliore in tessuti da piante PG e PMEI e che per essi non è necessario un pre-trattamento acido per ottenere una buona saccarificazione. Una possibile spiegazione è il contenuto ridotto di "zone di giunzione” per le peculiari caratteristiche di HGA in queste piante. Saccharification of PG and PMEI plants is accompanied by increased tissue maceration (see inserts in Fig. 1A-C). After a 24-hour incubation in the absence of cellulase, neither release of sugars nor maceration of the tissues are observed, proving that the reduction of the HGA content or its methylation does not in itself cause tissue disassembly and saccharification, but rather the ability of cellulases to degrade cellulose in intact tissue. Furthermore, the effectiveness of enzymatic hydrolysis on leaves pretreated with wild plant acids (WT) or transgenic (PG and PMEI) does not differ significantly (Fig. 1 D). It should be noted that, after 24 hours of saccharification, the effectiveness of enzymatic hydrolysis obtained with leaves pre-treated with acids from all plants is the same as that observed with non-pre-treated transgenic plants. No significant sugar release is obtained after the acid pre-treatment alone, regardless of the plants used. The results indicate that cellulose degradability is better in fabrics from PG and PMEI plants and that an acid pre-treatment is not required for them to achieve good saccharification. One possible explanation is the reduced content of "junction zones" for the unique characteristics of HGA in these plants.
E’ descritto che piante PG da tabacco hanno un contenuto ridotto di acido galatturonico (GalA) (23), che riduce la possibilità che si formino lunghe catene di HGA, necessarie per la formazione delle “zone di giunzione”. D’altra parte, sebbene le piante PMEI abbiano il medesimo contenuto di GalA delle selvatiche (WT), esse mostrano un aumentato grado di metilazione della pectina (24), che impedisce anch’esso la formazione delle “zone di giunzione”. It is described that PG tobacco plants have a reduced content of galacturonic acid (GalA) (23), which reduces the possibility of the formation of long chains of HGA, necessary for the formation of "junction zones". On the other hand, although PMEI plants have the same GalA content as wild (WT), they show an increased degree of methylation of pectin (24), which also prevents the formation of "junction areas".
Per verificare la presenza di regioni de-esterificate di HGA in piante PG e PMEI rispetto alle WT, sono usati: un anticorpo monoclonale PAM1 , che riconosce in maniera specifica grandi blocchi de-esterificati di HGA (almeno 30 unità continue di GalA) (25,26) e un anticorpo monoclonale JIM5, che si lega alla pectina a basso grado di metilazione (grado di metilesterificazione fino al 40%) (26). Si analizzano con saggi Immunologici (27) diluizioni seriali di polisaccaridi pectici arricchiti di poliuronidi (solidi chelanti agenti solubili, ChASS), estratti da foglie di piante di Arabidopsis WT o transgeniche come da Lionetti et al. (Lionetti, V. et al. To verify the presence of de-esterified regions of HGA in PG and PMEI plants with respect to WT, the following are used: a PAM1 monoclonal antibody, which specifically recognizes large de-esterified blocks of HGA (at least 30 continuous units of GalA) (25 , 26) and a JIM5 monoclonal antibody, which binds to low-methylation pectin (up to 40% methylesterification grade) (26). Serial dilutions of pectic polysaccharides enriched with polyuronides (soluble soluble chelating agents, ChASS), extracted from Arabidopsis WT plant leaves or transgenic as per Lionetti et al., Are analyzed with Immunological assays (27). (Lionetti, V. et al.
2007) . L’anticorpo PAM1 lega epitopi sia in piante PG che PMEI, ma in minore entità in piante WT, indicando che entrambi i trasformanti presentano una quantità ridotta di HGA demetilato. 2007). The PAM1 antibody binds epitopes in both PG and PMEI plants, but to a lesser extent in WT plants, indicating that both transformants have a reduced amount of demethylated HGA.
Anche l’anticorpo JIM5 lega epitopi in piante PG e PMEI, ma in minore entità in WT (Fig. 2), indicando la presenza di HGA a più elevata metilesterificazione. The JIM5 antibody also binds epitopes in PG and PMEI plants, but to a lesser extent in WT (Fig. 2), indicating the presence of HGA with higher methylesterification.
In conclusione, gli autori hanno dimostrato che la riduzione di HGA de-esterificato nelle pareti cellulari aumenta l'efficacia di idrolisi enzimatica nei tessuti vegetali. Tale modifica è vantaggiosamente usata per migliorare il processo di saccarificazione usato nella produzione di biocombustibili o di altri bioprodotti. Tale riduzione di HGA de-esterificato nelle pareti cellulari di tessuti vegetali può essere ottenuta con diverse modalità, quali la trasformazione genetica e l’ottenimento di piante transgeniche PG e/o PMEI; selezione di varianti naturali o indotte da mutagenesi, per la presenza di elevati livelli di PMEI endogeno. In conclusion, the authors demonstrated that the reduction of de-esterified HGA in cell walls increases the efficacy of enzymatic hydrolysis in plant tissues. This modification is advantageously used to improve the saccharification process used in the production of biofuels or other bioproducts. This reduction of de-esterified HGA in the cell walls of plant tissues can be obtained in various ways, such as genetic transformation and obtaining PG and / or PMEI transgenic plants; selection of natural variants or induced by mutagenesis, due to the presence of high levels of endogenous PMEI.
Le piante PMEI hanno una migliore saccarificazione e anche una resa di produzione di biomassa aumentata (circa 80% in più) (Tabella I) (17,24,28). PMEI plants have better saccharification and also increased biomass production yield (about 80% more) (Table I) (17,24,28).
Tabella I. Peso fresco (FW) e peso secco (DW) della rosetta di piante di Arabidopsis WT, PG e PMEI. Table I. Fresh weight (FW) and dry weight (DW) of the rosette of Arabidopsis WT, PG and PMEI plants.
I dati rappresentano la media ± s.e.m. di almeno 6 campioni indipendenti. Gli asterischi indicano differenze significative rispetto al WT secondo il test t di Student (P< 0.001). The data represent the mean ± s.e.m. of at least 6 independent samples. Asterisks indicate significant differences from WT according to Student's t-test (P <0.001).
Line Rosette FW (mg) DW /FW ratio (%) Line Rosette FW (mg) DW / FW ratio (%)
WT 137.9 ± 10 11.4 ± 1.8 WT 137.9 ± 10 11.4 ± 1.8
PG 81 .6 ± 2.9<***>9.9 ± 1.9 PG 81 .6 ± 2.9 <***> 9.9 ± 1.9
AtPMEI 253.4 1 18.3<***>11.2 ± 0.2 AtPMEI 253.4 1 18.3 <***> 11.2 ± 0.2
Inoltre le piante PMEI mostrano un’aumentata resistenza a patogeni microbici (24) e sono pertanto una fonte ideale di biocombustibile ma anche di altri prodotti commerciali. In addition, PMEI plants show increased resistance to microbial pathogens (24) and are therefore an ideal source of biofuel but also of other commercial products.
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