High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

Abstract

Background: Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose.

Results: In this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice straw.

Conclusion: Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for the large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in transgenic rice may also facilitate saccharification of cellulose in rice straw and significantly reduce the costs for hydrolytic enzymes.

Figure 1

Southern blot analysis of HptII and E1 genes. Genomic DNA was extracted from six independent T0 transgenic lines (#1 to #6) and wild type (WT) rice plants, digested with HindIII, separated by agarose gel electrophoresis, transferred to nylon membrane and hybridized with (A) HptII probe or (B) E1 probe. Note that E1 gene was not detected in transgenic line #4 and wild type rice plants.

Figure 2

Northern blot analysis of E1 gene transcript in the leaves. Twenty-five micrograms of total RNA, extracted from the leaves of six independent T0 transgenic lines (#1 to #6) and wild type (WT) rice plants, was loaded into each lane, transferred to nylon membrane after electrophoresis and probed with (A) E1 DNA or (B) stained for rRNA by EtBr. Note that no E1 gene transcript was detected in transgenic line #4 and wild type plants.

Figure 3

Tissue-specific expression of E1 gene, as analyzed by northern blot analysis. Twenty-five micrograms of total RNA, extracted from mature seed (MS), green floret (F), leaf (L), stem (S), and root (R) of T0 transgenic line #3-5 (one of the T0 plants of transgenic line #3) and wild type (WT) rice plants, was loaded into each lane, transferred to nylon membrane after electrophoresis and probed with (A) E1 DNA or (B) stained for rRNA by EtBr.

Figure 4

Western immunoblot analysis of E1 and Rubisco large subunit proteins in the leaf and different organs. Total soluble protein was extracted from the leaves of six independent T0 transgenic lines (#1 to #6) and wild type (WT) rice plants and from the mature seed (MS), green floret (F), leaf (L), stem (S) and root (R) of T0 transgenic line #3-5, separated by SDS-PAGE, transferred to polyvinylidene fluoride membrane and probed with polyclonal antibodies against (A) E1 or (B) Rubisco large subunit. Twenty micrograms of protein was loaded per lane. Note that E1 protein was not detected in the leaves of transgenic line #4 and wild type rice plants.

Figure 5

SDS-PAGE analysis of leaf soluble protein with or without prior heat pretreatment. Total leaf soluble protein was extracted from newly matured leaves of six independent T0 transgenic (#1 to #6) and wild type (WT) rice plants (A) without or (B) with prior heat treatment at 78°C for 45 min before electrophoretic separation by SDS-PAGE. Twenty micrograms of protein was loaded per lane. M: protein standards. After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250.

Figure 6

Specific activities of E1 enzyme in different organs. Specific activities of (A) E1 in the leaves of six independent T0 transgenic line (#1 to #6) and wild type (WT) rice plants, (B) in the mature seed (MS), green floret (F), leaf (L), stem (S) and root (R) of T0 transgenic line #3-5, and (C) in the leaves of some T1 plants of transgenic line #3-5. Enzyme activity was assayed using a fluorescence spectrophotometer by its ability to cleave 4-methylumbelliferyl β-D-cellobioside to produce the fluorophore, 4-methylumbelliferne with peak excitation wavelength at 365 nm and peak fluorescence at 455 nm. Data presented were means ± standard error (bar) from three to four replicates of measurement. The activity was measured at 65°C. Note both transgenic line #4 and wild type (WT) had minimal activities.

Figure 7

Zymogram analysis of E1 enzyme activity in the leaves. Total soluble protein was extracted from the leaves of six independent T0 transgenic lines (#1 to #6) and wild type (WT) rice plants and separated by SDS-PAGE in gel containing 0.1% carboxymethyl cellulose. After electrophoretic separation (20 μg/lane), the gel was washed and E1 activity stained in situ with Congo Red. The size of the clear zone indicates the degree of activity. Note that no E1 activity was detected in transgenic #4 and wild type plants. Purified E1 protein was obtained by heating (78°C) the leaf soluble protein extract of T0 transgenic line #3-5 for 45 min, and purified E1 protein: (A) 1 μg; (B) 2 μg; (C) 3 μg.

Figure 8

Release of sugars from rice straw during digestion with artificial saliva or cultured cow gastric fluid. The contents of (A) reducing sugars, (B) sucrose, (C) glucose and (D) fructose from the straws of wild type (WT) and homozygous E1 transgenic rice plants (derived from line #3-5) after incubation with AS or cultured CGF at 39°C for 1 h, 39°C for 2 h, or 39°C for 1 h plus 81°C for 1 h. WT/AS: WT rice straw incubated with AS; T/AS: transgenic rice straw incubated with AS; WT/CGF: WT rice straw incubated with CGF; T/CGF: transgenic rice straw incubated with CGF. Rice straw was air dried under the sun and stored at room temperature for two years. Data presented represent the means ± standard error of three replicates of measurement. AS: artificial saliva; CGF: cow gastric fluid; T: transgenic; WT: wildtype.