Expression of Acidothermus cellulolyticus E1 endo-β-1,4-glucanase catalytic domain in transplastomic tobacco

Plant Biotechnology Journal (2009) 7, pp. 527–536

doi: 10.1111/j.1467-7652.2009.00421.x

Expression of Acidothermus cellulolyticus E1 endo-β-1,4-glucanase catalytic domain in transplastomic tobacco T. Original Endoglucanase Ziegelhoffer Article et expression al. Blackwell Oxford, Plant PBI © 1467-7652 1467-7644 XXX 2009 Biotechnology UK Blackwell Publishing Publishing Journal Ltd Ltd in transplastomic tobacco

Thomas Ziegelhoffer*, John A. Raasch and Sandra Austin-Phillips Biotechnology Center, University of Wisconsin-Madison, 425 Henry Mall, Madison, WI 53706, USA

Received 24 November 2008; revised 27 February 2009; accepted 1 April 2009. *Correspondence (fax 608 262-6748, e-mail [email protected])

Summary As part of an effort to develop transgenic plants as a system for the production of lignocellulose-degrading enzymes, we evaluated the production of the endo-β-1,4-glucanase E1 catalytic domain (E1cd) of Acidothermus cellulolyticus in transplastomic tobacco. In an attempt to increase the translation efficiency of the E1cd cassette, various lengths of the N-terminus of the psbA gene product were fused to the E1cd protein. The psbA gene of the plastid genome encodes the D1 polypeptide of photosystem II and is known to encode an efficiently translated mRNA. Experiments in an Escherichia coli expression system indicated that the fusion of short (10–22 amino acid) segments of D1 to E1cd resulted in modest increases in E1cd abundance and were compatible with E1cd activity. Plastid expression cassettes encoding unmodified E1cd and a 10-amino-acid D1 fusion (10nE1cd) were used to generate transplastomic tobacco plants. Expression of the E1cd open reading frame in transplastomic tobacco resulted in very low levels of the enzyme. The transplastomic plants accumulated a high level of E1cd mRNA, however, indicating that post-transcriptional processes were probably limiting the production of recombinant protein. The accumulation of 10nE1cd in transplastomic tobacco was approximately 200-fold higher than that of unmodified E1cd, yielding 10nE1cd in excess of 12% of total soluble protein in the extracts of the lower leaves. Most importantly,

Keyords: cellulase, transgenic plant,

the active recombinant enzyme was recovered very easily and efficiently from dried plant

plastid transformation, bioenergy.

material and constituted as much as 0.3% of the dry weight of leaf tissue.

Introduction The efficient large-scale conversion of lignocellulosic materials to ethanol could substantially reduce US dependence on petroleum, whilst utilizing biomass resources that are readily available under current forestry and agricultural practices (Perlack et al., 2005). The emergence of bioenergy as a significant part of a future renewable energy sector will require the optimization of several technologies for its successful implementation (Gressel, 2008; Yuan et al., 2008). One such technology, the conversion of lignocellulosic materials to simple sugars for fermentation to fuel ethanol or other feedstock chemicals, has been identified as a major barrier to the economical production of cellulosic ethanol (US © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd

Department of Energy, 2006; Himmel et al., 2007). This is a result, in part, of the fact that lignocellulosic materials are a composite of the biopolymers cellulose, hemicellulose and lignin. Because these biopolymers are chemically distinct, yet covalently interconnected, physical pretreatment is an essential first step in enzyme-mediated lignocellulose deconstruction (Mosier et al., 2005). Even after pretreatment, cellulose fibres are recalcitrant to the action of any single enzyme activity. A minimal set of enzymes for cellulose deconstruction includes endoglucanase, exoglucanase and β- D -glucosidase activities (Zhang et al ., 2006). It is also apparent that activities in addition to glycoside hydrolases (expansin-like proteins, for example) may also play a role in an optimized enzyme mixture (Saloheimo et al., 2002).

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A significant fraction of the cost of the cellulose to ethanol conversion process is the cost of the enzymes used in the depolymerization of cellulose. One way to reduce this cost is to increase the catalytic efficiency of individual enzymes and enzyme mixtures (Rosgaard et al., 2006; Berlin et al., 2007), thereby reducing the enzyme load required for a given degree of hydrolysis. Another way to lower the cost of enzymes is to reduce their cost of production. Towards that end, a number of research groups have investigated the practicality of producing various cellulases in crop plants (Dai et al., 1999, 2000, 2005; Ziegelhoffer et al., 1999, 2001; Ziegler et al., 2000; Jin et al., 2003; Teymouri et al., 2004; Biswas et al., 2006; Hood et al., 2007; Yu et al., 2007; Gray et al., 2009). As an alternative to microbial production of enzymes in fermenters, crop plants offer the potential advantages of

1994). In the current work, we describe the construction and analysis of transplastomic tobacco plants expressing the E1 catalytic domain (E1cd). We show that an E1cd fusion product bearing the N-terminal amino acids of a highly expressed plastid protein accumulates to very high levels in transplastomic plants.

rapid scalability and reduced capital investment. Cellulolytic enzymes could be produced directly in biomass crops, with enzyme extraction occurring prior to the pretreatment step. In addition, the expression of these enzymes in biomass crops could potentially yield biomass with more favourable properties for bioprocessing (Sticklen, 2006, 2007; Rubin, 2008). Alternatively, enzymes could be produced in a dedicated enzyme crop such as alfalfa (Austin et al., 1995; Ullah et al., 2002), an extract of which would then be added to pretreated biomass. The production of cellulose-degrading enzymes in plants is part of a larger effort to expand the use of plants as production systems for various non-food products (Hood, 2002; Twyman et al., 2003; Teli and Timko, 2004; Howard and Hood, 2007; Streatfield, 2007). In the production of heterologous enzymes in plants, high levels of accumulation are important to achieve an economically viable process. One strategy to accomplish this that has met with considerable success is plastid transformation. Unlike nuclear transformation, plastid transformation results in thousands of transgene copies per cell. This, in part, contributes to the high level of heterologous protein production observed in some transplastomic plants: more than 70% of total soluble protein (TSP) in the case of the antibiotic protein lysin (Oey et al., 2009) and frequently between 5% and 20% of TSP (reviewed in Maliga, 2004; Koya and Daniell, 2005; Bock, 2007; Lutz et al., 2007; Verma and Daniell, 2007). In previous work, we have investigated the effects of subcellular targeting and protein truncation on the accumulation level of the E1 endo-1,4-β-D-glucanase (endoglucanase)

Kuroda and Maliga, 2001; Ye et al., 2001; Gray et al., 2009). This prompted us to investigate the effect of various fusions of the endogenous plastid D1 N-terminal sequence to the E1cd coding sequence. The D1 polypeptide of photosystem II, encoded by the plastid psbA gene, was chosen because of its high apparent translation efficiency. In addition to lightregulated initiation of translation via 5′ untranslated region (UTR) sequences (Staub and Maliga, 1994; Zou et al., 2003), the psbA coding sequence shows evidence of codon bias favouring maximum translation rate (Morton, 1998). We sought to determine whether portions of the 5′ coding sequence of psbA could confer enhanced accumulation to a recombinant product in transplastomic tobacco. A series of N-terminal fusions of increasing length was tested in E. coli using the pET vector system (Rosenberg et al., 1987). The E1cd plastid construct incorporates an NcoI site at the start of the mature coding sequence. This NcoI site was utilized in the creation of a set of fragments encoding up to

of Acidothermus cellulolyticus in nuclear transformants of tobacco (Ziegelhoffer et al., 2001). This cellulase was chosen primarily because of its high heat stability (Topt ~ 80 °C) and its high activity in the hydrolysis of the synthetic substrate 4-methylumbelliferyl-β- D -cellobioside (MUC) (Baker et al.,

Results Fusion of D1 N-terminal sequences to E1cd and expression in Escherichia coli A number of authors have made the observation that the N-terminal coding sequence of a protein can profoundly effect its accumulation level in plastids (Morton, 1998;

36 amino acids of the D1 polypeptide. The 5′ and 3′ ends of each fragment incorporated a BspHI site (compatible for ligation with NcoI-cleaved DNA) and NcoI site, respectively. In this way, vectors encoding fusions of 10, 15, 22 and 36 amino acids were constructed (Figure 1a). Each plasmid was used to transform E. coli BL21 (DE3) pLysS, and the resulting strains were tested for E1cd accumulation on induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). Fusion proteins incorporating 10, 15 and 22 N-terminal amino acids of D1 accumulated to higher levels than unmodified E1cd, whereas the 36-amino-acid fusion showed a significant decrease in accumulation (Figure 1b). A gradual increase in apparent molecular weight correlated with increased fusion length. Activity measurements of crude extracts reflected the increase in apparent abundance of E1cd when fused to 10 amino acids of the D1 N-terminus (10nE1cd). Longer fusions

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Figure 1 D1 N-terminal fusions in Escherichia coli. A set of fusion constructs was prepared using the E. coli pET expression system. (a) Fusions incorporating 10–36 amino acids of the D1 amino terminus are aligned with the D1 protein sequence. The methionine corresponding to the initiating amino acid in the ‘unmodified’ endo-β-1,4-glucanase E1 catalytic domain open reading frame (E1cd ORF) is indicated in bold. It should be noted that Met36 of the D1 polypeptide starts the first of five transmembrane α-helices (indicated by a broken underline). (b) Extracts of induced cultures were analysed by Western blot (5 μg total protein per sample). In each case, the transforming plasmid is indicated above the appropriate lane. Monoclonal antibody directed against E1cd was used to detect the recombinant product. An asterisk indicates the expected mobility of E1cd. E1 holoenzyme (5 ng) is included as a positive control. (c) 4-Methylumbelliferyl-β-D-cellobiosidase (MUCase) activity measurements of E1cd extracts revealed negative effects of fusions longer than 10 amino acids. The identity of the transforming plasmid is indicated on the vertical axis. The assay measures the release of 4-methylumbelliferone (4-MU) from the fluorogenic substrate MUC. Extracts were normalized on the basis of total protein content. Values represent the average of duplicate determinations.

showed progressively less activity, suggesting the possibility that they interfered with activity (Figure 1c). Activity assays carried out after a 5 min heat treatment of extracts at 70 °C showed no difference from untreated samples, supporting the conclusion that the stability of fusions up to 22 amino acids was similar to that of unmodified E1cd (data not shown). Very little activity was measured for the 36nE1cd product.

Plastid transformation of tobacco with E1cd constructs For the generation of transplastomic tobacco, we constructed plastid transformation vectors in which the sequence encoding mature E1cd was placed between the trnI and trnA genes of the rRNA operon (Figure 2). Sequences for both E1cd and 10nE1cd were used to generate expression cassettes. The expression cassette was inserted in tandem with an aadA cassette (conferring spectinomycin resistance). Preliminary experiments indicated that a promoterless aadA cassette was efficiently incorporated into the plastid genome at this site, conferring spectinomycin resistance and ultimately

Figure 2 Schematic representation of transgene insertion into the tobacco rrn operon of the plastid genome. Insertion of the aadA + E1cd/10nE1cd tandem cassette into the intergenic region between trnI and trnA is indicated. Transgene cassettes incorporate the ribosome binding site of T7 gene 10 and the 3′ untransformed region (UTR) of Medicago sativa psbA, as indicated. Insertion was via SphI sites at the ends of the cassettes and an SphI site between trnI and trnA. The subsequent vector, containing approximately 2 kbp on either side of the insertion site, was used to transform tobacco by microparticle bombardment as described. The direction of transcription is from left to right. XmnI sites used for Southern analysis are indicated. Not to scale.

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yielding homoplastomic plants. Both tandem orientations (E1cd > aadA, aadA > E1cd) were used to generate transplastomic tobacco plants by biolistic transformation. Regardless of orientation, the expression levels in the leaves of transplastomic E1cd plants reached a maximum of approximately 0.05% TSP. This expression level is much lower than the maximum level previously documented in nuclear transformed plants, in which E1cd was targeted to the plastid via the RuBisCo small subunit targeting peptide and expressed under the strong constitutive cauliflower mosaic virus (CaMV) 35S promoter (Ziegelhoffer et al., 2001). By contrast, the 10nE1cd construct yielded plants with expression levels approximately 100 times greater than the corresponding E1cd construct in primary transformants. Transplastomic plants were subjected to two cycles of recurrent selection, yielding regenerants that were homoplastomic for the transgenes, as indicated by the shift in mobility of the associated XmnI band from 2.3 to 4.5 kbp (Figure 3). Transplastomic plants were fertile, with all progeny exhibiting a specR.phenotype. Seed viability in transplastomic plants was indistinguishable from that of Petit Havana control plants. 10nE1cd plants were somewhat slower growing than untransformed controls (Figure 4), such that flowering typically occurred approximately 1 week later in 10nE1cd plants. By contrast, transplastomic E1cd plants showed a markedly decreased growth rate. The growth rate difference persisted throughout development, resulting in an approximate doubling of the time to flowering for E1cd plants compared with 10nE1cd or the wild-type. Mature plant parts were used to prepare extracts to be analysed for MUCase activity and total protein concentration. All tissues sampled showed MUCase activity, corresponding to E1cd concentrations up to nearly 0.06% of TSP in upper stem sections (Figure 5a). In contrast, 10nE1cd was present at much higher levels in all tissues examined, with levels up to 12% of TSP in lower leaves (Figure 5b). Both E1cd and 10nE1cd produced in transplastomic plants showed the stability in crude plant extracts typical for this enzyme. A comparison of total RNA isolated from transplastomic plants by Northern blot showed that mRNA levels were very similar in E1cd and 10nE1cd plants, despite several orders of magnitude difference in protein levels. However, 10nE1cd plants showed greatly reduced levels of dicistronic RNA containing both E1cd and aadA sequences (Figure 6). In addition, 10nE1cd plants had higher levels of truncated (less than full-length) mRNA. In both E1cd and 10nE1cd plants, polycistronic mRNA was much more abundant than monocistronic mRNA. Direct comparison of transplastomic mRNA levels with those of E1cd-expressing nuclear transformants previously documented (Ziegelhoffer

Figure 3 Southern blot of genomic DNA from transplastomic plants indicates that they are homoplasmic. DNA isolated from seedlings of PT2074 (E1cd), PT2149 and PT2151 (10nE1cd) and Petit Havana control was digested with XmnI, fractionated on 1% agarose and transferred to Hybond N+ membrane. Chemiluminescent detection of hybridizing bands (Gene Images System, Amersham Biosciences) revealed the expected shift from 2.3 kbp (*) to approximately 4.5 kbp (**) in transplastomic plants. The band of approximately 2.7 kbp (>) present in all plant-derived samples is a consequence of probe hybridization to the XmnI fragment incorporating the 5′ end of the 16S RNA gene and rrn promoter (see Figure 2). The XmnI-digested plasmid control pP3.27-1 (targeting vector) yields a 2.3-kbp band corresponding to the wild-type insertion site.

Figure 4 Growth of transplastomic plants in pots. Transplastomic and control seeds were sown in soil-free potting mix in 4-in (10 cm) pots. After approximately 2 weeks, seedlings were thinned to one plant per pot. Plants were photographed approximately 5 weeks later (7 weeks after planting). PH, Petit Havana control; E1cd, PT2074 (front) and PT2076; 10nE1cd, PT2149 (front) and PT2151.

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Figure 6 Northern analysis of RNA isolated from transplastomic tobacco. Total RNA was isolated from tobacco plants and resolved on a denaturing agarose gel (2 μg total RNA per well). RNA was transferred to a nylon membrane for Northern blot analysis using a non-radioactive labelling and detection kit. The blot was probed with both E1cd and aadA probes, as indicated. Samples: (1) PT2080, transplastomic E1cd; (2) PT2149, transplastomic 10nE1cd; (3) PH, cv. ‘Petit Havana’. Asterisk indicates the mobility of presumptive dicistronic E1cd/aadA mRNA. > indicates the mobility of E1-specific monocistronic mRNA.

Figure 5 E1cd/10nE1cd activity recovered from various plant parts. Samples were removed from flowering plants grown in potting mix (PT2080, E1cd; PT2149, 10nE1cd). Extracts were prepared and assayed for protein content (Bio-Rad assay) and 4-methylumbelliferyl-β-Dcellobiosidase (MUCase) activity. E1cd abundance is expressed as a percentage of total soluble protein (TSP) extracted under these conditions and is based on enzymatic activity. Control extracts were used for background subtraction. Upper and lower leaves are the topmost and lowest non-senescent leaves, respectively. Upper and lower stem samples were removed from internode sections immediately below the inflorescence and just above soil level, respectively. Flower samples consisted of the distal 1 cm of the corolla. Values shown are the average of duplicate determinations.

et al., 2001) showed that even monocistronic message in plastid transformants was much more abundant than the corresponding mRNA in nuclear transformants (data not shown).

a forced air oven or kept on ice. The next day, distilled H2O was added to the dried sample to return it to its fresh weight. Both the dried and iced samples were then ground to homogeneity and analysed for extractable protein and assayed for MUCase activity. As expected, drying reduced the recoverable TSP significantly, ranging from 30.5% recovery in roots to 10.8% in upper stem sections (Figure 7a). MUCase activity remained highly soluble, however, ranging from 93% recovery in dried root samples to 63% recovery in young leaves. When plotted as a percentage of dry matter, it was evident that much of the E1cd activity was present in leaves, with E1cd representing about 0.3% of dry matter in older leaves (Figure 7b). Because stem material comprises the bulk of the dry matter in the above-ground portion of the plant, a significant fraction of the total E1cd is also contributed by this fraction. The extraordinary solubility of E1cd in dried tissue was clearly demonstrated by sodium dodecylsulphatepolyacrylamide gel electrophoresis (SDS-PAGE) fractionation of extracts of dried leaves (Figure 8), with E1cd as the single most abundant protein in such extracts.

Discussion 10nE1cd recovery from dried plant tissue To assess the recoverability of 10nE1cd from transplastomic tobacco, samples were removed and either dried at 55 °C in

Crop plants have been successfully used to produce proteins with utility in healthcare, animal agriculture and industrial processes (Kusnadi et al., 1997; Teli and Timko, 2004; Verma

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Figure 7 10nE1cd activity is efficiently recovered from dried plant material and represents a significant fraction of dry weight. Samples were removed from flowering plants grown in potting mix. Equivalent samples were placed in duplicate microcentrifuge tubes and either placed on ice or dried at 55 °C for 20 h. For the dried samples, 1 μL of water per milligram sample lost on drying was added prior to extraction. Extracts were prepared and assayed for protein content (Bio-Rad assay) and 4-methylumbelliferyl-β-D-cellobiosidase (MUCase) activity. (a) MUCase recovery and total protein recovery were determined for several tissue samples. Values are given as a percentage of the value obtained from duplicate samples stored on ice. (No significant difference was observed in either protein or MUCase recovery between fresh samples and those stored on ice overnight). Upper and lower stem sections (approximately 2 cm long) were removed either immediately below the inflorescence or immediately above soil level, respectively. Upper and lower leaves were the topmost and lowest non-senescent leaves, respectively. (b) The quantity of 10nE1cd (MUCase activity) extracted from fresh or dried tissues is reported as a percentage of total dry matter. For this analysis, dry matter is defined as the sample mass remaining after equilibration at 55 °C at a relative humidity of approximately 15% or less. The values depicted represent the average of duplicate measurements.

Figure 8 10nE1cd is the predominant protein species in extracts of dried PT2149 leaves. Extracts from control Petit Havana and 10nE1cd-expressing PT2149 leaves were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue. Each lane contains extract corresponding to 1 mg of fresh weight. Samples of leaves were collected from each plant, corresponding to leaves 2, 6 and 10 counting from the base of the inflorescence (T, top; M, middle; B, bottom). Each leaf sample was divided in half and processed as fresh or dried sample as described above. E1cd control was included as indicated (ng pure protein). The asterisk indicates the mobility of intact E1cd/10nE1cd (the two species do not differ significantly in mobility under the separation conditions used).

and Daniell, 2007). The practical viability of crop plants as ‘bioreactors’ (Goddijn and Pen, 1995) depends, to a considerable extent, on the level of product obtained. In an effort to increase the level of recombinant cellulase produced in transgenic plants, we produced transplastomic tobacco plants expressing E1cd of A. cellulolyticus. N-terminal modification to enhance protein accumulation (through enhanced translation efficiency) has been used previously in transplastomic systems (Khan and Maliga, 1999; Ye et al., 2001), including cellulase expression (Yu et al., 2007; Gray et al., 2009). We chose to test the effect

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of fusing the 5′ portion of the very efficiently translated psbA open reading frame (ORF) to the E1cd coding sequence. Initial experiments utilizing the E. coli pET expression system showed that fusions of 30–66 nucleotides (encoding the N-terminal 10–22 amino acids of the photosystem II D1 protein) resulted in enzymatically active fusion proteins, although fusions of 15 and 22 amino acids resulted in lower apparent E1cd activity. The 10-aminoacid fusion (10nE1cd) was selected for expression in transplastomic plants. Although we used Medicago truncatula psbA sequences for these studies, functional conservation of psbA genes suggests that psbA sequences from other species would behave similarly. For example, the N-terminal 36 amino acids of D1 from tobacco and M. truncatula differ at only two positions: 9 (glutamic acid and aspartic acid, respectively) and 12 (serine and aspartic acid, respectively). We constructed a vector for insertion of transgenes into the plastid ribosomal RNA operon based on published work showing the efficacy of this approach (De Cosa et al., 2001). Plastid expression cassettes for the selectable marker (aadA) and E1cd were designed using elements similar to those that have been shown to function well in transplastomic tobacco (Svab and Maliga, 1993; Ye et al., 2001). Our transplastomic tobacco lines differed from those in most other published reports in that this set of constructs lacked internal promoter sequences, taking advantage of the strong Prrn promoter to drive the expression of transgene cassettes. Recent reports have shown the effectiveness of read-through transcription from the Prrn promoter in the expression of transgenes (Chakrabarti et al., 2006; Gray et al., 2009). It has been observed previously that P rrn-driven expression generates polycistronic RNAs incorporating transgenes when the constructs are inserted within the rRNA operon, even when the transgene constructs incorporate their own separate promoters (Quesada-Vargas et al., 2005). Indeed, we observed abundant E1cd-specific mRNA in transplastomic tobacco plants. Both E1cd and 10nE1cd transformations yielded homoplastomic plants, based on Southern blot data. Northern blots showed the presence, in E1cd transplastomic plants, of a very abundant mRNA species corresponding to dicistronic message incorporating the coding sequences of E1cd and aadA. This mRNA species was not evident in 10nE1cd plants. This major difference between E1cd and 10nE1cd plants suggests the possibility that dicistronic mRNA may be responsible for the slow growth observed in E1cd plants. Further experiments are required to determine whether this is the case. Conversely, the presence of similar levels of most other E1cd-containing

transcripts in both E1cd and 10nE1cd plants implies that the translation efficiency is probably enhanced in10nE1cd plants. Protein stability does not appear to play a role, as the E1cd produced in transplastomic E1cd plants, although not very abundant, is very resistant to activity loss in crude plant extracts. The 10nE1cd cassette yielded very high levels of enzyme accumulation in transplastomic tobacco (in excess of 12% TSP or 0.3% total dry matter in older leaves). Significant accumulation of E1cd activity was observed in all tissues examined, with the highest levels present in leaves and the lowest levels in roots (consistent with the chloroplast localization of the recombinant product). In the analysis of leaves of varying age, E1cd activity was relatively constant as a function of fresh weight. Older leaves yielded the highest levels on a protein basis because of the extensive recycling of endogenous proteins in senescent leaves. As observed in previous work, E1cd is extraordinarily stable to proteolysis in crude plant extracts and is apparently able to evade the protein recycling mechanisms of the plant. Protease-resistant recombinant lysin showed a similar pattern of stability in ageing leaves of transplastomic tobacco (Oey et al., 2009). The growth rate of 10nE1cd transplastomics was slightly slower than that of Petit Havana controls, with some chlorotic patches usually evident on lower leaves. It is tempting to speculate that the slightly slower growth observed for 10nE1cd transplastomics could be a consequence of resource allocation, as the abundance of the recombinant product rivals that of the RuBisCo large subunit, generally the most abundant protein in green plant tissues. It seems likely that the relatively low activity of E1cd at plant growth temperature and the inaccessibility of its substrate both play a role in minimizing phenotypic effects. One of the key considerations for the production of industrial or feed enzymes in crop plants is the recovery of the recombinant product from the transgenic plant after harvest. Ideally, the recombinant product should: (i) be produced in high yield; (ii) be stable in dried plant material; and (iii) be readily recovered using a simple low-cost extraction procedure. 10nE1cd produced in transplastomic tobacco satisfies all three criteria. Our demonstration of high endoglucanase expression levels in transplastomic tobacco, and recent work published by others showing similarly high expression levels for several other cellulase enzymes (Hood et al., 2007; Yu et al., 2007; Gray et al., 2009), supports a role for plant expression systems in the production of enzymes for use in the conversion of lignocellulosic biomass.

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Experimental procedures Vector construction The plasmid pP3.13-7 (carrying 3662 bp of tobacco plastid DNA including the 3′ part of the 16S RNA gene, the trnA and trnI genes, as well as the 5′ end of the 23S RNA gene) was constructed by polymerase chain reaction (PCR) amplification of the region using Nicotiana tabacum (cv. Wisconsin 38) genomic DNA and the oligonucleotides 5′-AAGAATGAAACTCAAAGGAATTG-3′ and 5′-GTCATATCTAG TATTCAGAGTTT-3′. The amplified product was cloned into pSTBlue-1 (Novagen/EMD Biosciences, Inc., La Jolla, CA, USA). KpnI digestion and re-ligation of pP3.13-7 removed 85 bp, including an SphI site in the vector multiple cloning region, yielding pP3.27-1. DNA sequencing of the insert revealed a single base substitution (A to G at nucleotide 645 of the 23S RNA gene), 263 bp from the end of the clone. pP3.27-1 is the basis for all targeting vectors used in this work. A recombinant aadA cassette conferring spectinomycin resistance combines sequence elements from bacteriophage T7 (Ye et al., 2001) and M. sativa (Aldrich et al., 1988; Svab and Maliga, 1993; Hajdukiewicz et al., 1994). The aadA gene in plasmid pPZP211 (Hajdukiewicz et al., 1994) was modified by the addition of NcoI and SacI at the 5′ and 3′ ends of the ORF, respectively. The recombinant ORF was generated by PCR using oligonucleotides 5′-GAGTCGACCATGGCGGAAGCGGT GATTCGCCGAA-3′ and 5′-ACCTCGAGAATAAACGGCTGGTAGAAC CA-3′. A short (39-nucleotide) XbaI to NcoI fragment of pET-14b (Novagen/EMD Biosciences), comprising the T7 ribosome binding site, was fused to the 5′ end of the aadA ORF. The 3′ UTR of M. sativa psbA was cloned from M. sativa RSY27 (Bingham, 1991) genomic DNA by PCR using the oligonucleotides 5′-GAGCGCGGTTTAAAAAAAG GATACGA-3′ and 5′-GTATACAGAAAAAGACGACTA-3′. These primers incorporate sites for SacI and AccI, respectively. The final aadA cassette is flanked by SphI sites at both ends, facilitating cloning into the unique SphI site of pP3.27-1. Several E1cd cassettes were constructed by modification of an existing clone (Ziegelhoffer et al., 2001). The construct referred to as ‘E1cd’ was modified by the addition of an NcoI site and ATG codon at the site of leader peptide cleavage in the wild-type preprotein, resulting in the addition of an N-terminal methionine in place of the signal peptide. PCR amplification with oligonucleotide SacE1cd (Ziegelhoffer et al., 2001) and oligonucleotide 5′-CCATGGCGGGC GGCGGCTATTG-3′ introduced an NcoI site at the 5′ end of the ORF, facilitating its fusion to the T7 gene 10 ribosomal binding site. In addition, the AGC codon specifying Ser48 of mature E1 was modified to AGT by site-directed mutagenesis (oligonucleotides 5′-TACCG CAGTATGCTCGAC-3′ and 5′-TCGAGCATACTGCGGTAG-3′), removing an internal SphI site. A set of 5′ fusions incorporating varying lengths of the psbA 5′ sequence was constructed by PCR amplification using the oligonucleotides 5′-CCATGGAATCGCGTCTCTCTAAAATTG-3′ (10nE1cd fusion), 5′-CCATGGACCATAGGTTTTCGCTATCGC-3′ (15nE1cd fusion), 5′-CCATGGTTATCCAGTTACAGAAG-3′ (22nE1cd fusion), 5′-CCATGGAAACACCAAACCATCCAATG-3′ (36nE1cd fusion) and the oligonucleotide 5′-GATTTTATCATGACTGCAATTTTA-3′ (BspHI site at start codon). In each case, M. truncatula cv. Jemalong (genotype 2HA) DNA was used as template. Because BspHI is compatible with NcoI in ligations, resulting constructs retain a single NcoI at the fusion junction, facilitating subsequent in-frame fusions of other ORFs with the 5′ psbA sequences. Both the E1cd and 10nE1cd ORFs were used to construct cassettes analogous to the aadA cassette

described above (T7 gene 10 ribosomal binding site, M. sativa psbA 3′ UTR). A complete set of E1cd fusions was cloned into pET-14b for E. coli expression studies. These plasmids were used to transform BL21 (DE3) pLysS (Novagen/EMD Biosciences) competent cells, as recommended by the manufacturer. IPTG induction was carried out as recommended by the manufacturer. Cells were harvested and concentrated by centrifugation 3 h after induction.

Plastid transformation Methods for transformation and selection of transgenic plants are essentially those described by others (Svab and Maliga, 1993). Nicotiana tabacum cv. Petit Havana plants were grown aseptically on MS (Murashige & Skoog) basal salts plus 30 g/l sucrose, 0.6% agar. Leaf explants (approximately 1 cm2) were placed on RMOP medium and bombarded with tungsten microcarriers (1.1 μm average particle size, Bio-Rad M-17) using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA, USA). Microcarriers were coated with DNA as recommended by the manufacturer. The target shelf was 9 cm from the microcarrier launch assembly and 900-psi rupture discs were used. Explants were moved to RMOP + spectinomycin (500 μg/mL) 2–3 days after bombardment. Resistant shoots were moved to the same medium and rooted on Petite Havana Growth medium. Two additional cycles of regeneration were carried out to ensure homoplasmy. DNA was isolated from plants using the method of Fulton et al. (1995). DNA was analysed by a combination of PCR and Southern blotting (Gene Images system, Amersham/GE Healthcare UK Limited, Little Chalfont Buckinghamshire HP7 9NA UK). The probe used in Southern blots was a 1.7-kbp PCR product generated using the 16S primer described above (5′-AAGAATGAAACTCAAAGGAATTG3′) and a primer hybridizing downstream of the SphI insertion site (5′-CCGG TACCAACTGAGCTATATCC-3′). Plants were then transferred to soil-free potting mix and grown to maturity for seed collection and analysis (24 °C, 15 h light, 9 h dark).

Expression analysis Transcript abundance was estimated by non-radioactive Northern blot (Amersham Gene Images AlkPhos Direct Labelling and Detection System, GE Healthcare). Denaturing formaldehyde-agarose gel electrophoresis and transfer to Hybond N+ membrane (Amersham, GE Healthcare) were carried out as recommended by the manufacturer. Total leaf RNA was isolated from growth chamber plants using the Qiagen RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). DNA probes were generated by PCR amplification. For the E1cd probe, the primers described above (see ‘Vector construction’ section) where used to amplify a fragment of approximately 1.1 kb. For the aadA probe, the aadA-specific primers described above (see ‘Vector construction’ section) were used to amplify a fragment of approximately 0.8 kbp using the plasmid pP3.27-1 as template. Protein extracts of E. coli cells were prepared using a detergent concentrate (PopCulture Reagent, Novagen/EMD Biosciences, Inc.). Protein extracts of tobacco tissue samples were extracted as described previously (Ziegelhoffer et al., 2001), with the exception that the ammonium sulphate precipitation step was excluded. Proteins were fractionated by SDS-PAGE (MiniPROTEAN II apparatus, Bio-Rad Laboratories) using 10–15% (w/v) acrylamide gradient gels. Western blots were prepared as described previously. Activity assay of E1cd utilized the model substrate MUC and was carried out as described

© 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 527–536

Endoglucanase expression in transplastomic tobacco 535

previously (Ziegelhoffer et al., 2001), except that the reaction temperature was 55 °C. All values presented are the average of duplicate determinations, with no more than 10% variation at any data point.

Acknowledgements We acknowledge the generosity of Steven R. Thomas (formerly at the National Renewable Energy Laboratory, Golden, CO, USA, currently Ceres, Inc., Thousand Oaks, CA, USA), who provided us with the E1 gene, purified E1 holoenzyme and catalytic domain, and anti-E1 antibody. This work was supported by the National Research Initiative of the US Department of Agriculture Cooperative State Research, Education and Extension Service (grant number 2005–02650).

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