The maize α-zein promoter can be utilized as a strong inducer of cellulase enzyme expression in maize kernels

Transgenic Research - Expression of recombinant proteins in plants is a technology for producing vaccines, pharmaceuticals and industrial enzymes. For the past several years, we have produced...     

Heterologous expression of cellobiohydrolase II (Cel6A) in maize endosperm

The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.     

Development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli

Summary Advances in the development of subunit vaccines and in the production of foreign proteins in plants together offer the prospect of stable and inexpensive vaccine delivery systems. Various bacterial and viral proteins stably produced in plants have been shown to elicit immune responses in feeding trials. We have extended this approach by using Zea mays as the plant production system. Corn has several advantages as a vaccine delivery vehicle, most notably established technologies to generate transgenic plants, to optimize traits through breeding and to process the seed into a palatable form. Here we report on the production in corn seed of the GM₁ receptor binding (B) subunit of the heat-labile toxin (Lt) from enterotoxigenic strains of Escherichia coli. Versions of the Lt-B gene were synthesized to give optimum codon usage for corn and to target the protein to either the cell surface or the cytoplasm. These synthetic genes were fused to a strong promoter and transformed into corn. Lt-B was highly expressed in corn seed at up to 1.8% of the total soluble protein and this was further increased approximately five-fold through plant breeding. As in E. coli. Lt-B produced in corn forms a functional pentamer that can bind to the GM₁ receptor. Furthermore, Lt-B pentamer stored in corn seed is much more resistant to heat than is the pure protein, allowing the transgenic corn to be readily processed into an edible form. This work demonstrates the potential of using products derived from transgenic corn seed as delivery vehicles for subunit vaccines.     

Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps.

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.     

Synthetic Lignin Mineralization by Ceriporiopsis subvermispora Is Inhibited by an Increase in the pH of the Cultures Resulting from Fungal Growth

(sup14)C-synthetic lignin mineralization by the basidiomycete Ceriporiopsis subvermispora occurs at the highest rate (about 30% after 29 days) in liquid cultures containing 1% glucose and a growth-limiting amount (1 mM) of ammonium tartrate. The titers of manganese peroxidase (MnP) and laccase are lower in these cultures than in cultures containing 1% glucose and 10 mM ammonium tartrate, where the extent of lignin mineralization in the same period is only about 15%. The inverse correlation between enzyme activity and lignin mineralization is also observed when ammonium tartrate is replaced by ammonium chloride or Casamino Acids as the source of nitrogen. This phenomenon can be explained by a gradual increase in the pH of the medium that takes place only in the cultures with high nitrogen concentrations. Supporting this finding, when cultures with 1 mM ammonium tartrate were grown at different pHs, (sup14)CO(inf2) evolved more rapidly from those with pH values near the optimum for MnP activity. On the other hand, (sup14)CO(inf2) evolution from cultures containing 1% glucose supplemented with 1 mM ammonium tartrate plus 9 mM sodium tartrate was as low as that from cultures with a high ammonium tartrate concentration. Since the changes in the pH of these cultures were not as pronounced as those in cultures containing high nitrogen concentrations, tartrate itself may also be contributing to limit the extent of lignin mineralization. Considering that pH instability seems to constitute a common feature of fungal cultures, precautions must be taken to avoid underestimation of their ligninolytic efficiencies.     

A method for transient expression in maize endosperm

The ability to screen the functionality of gene constructs in a transient system of appropriate tissues informs the researcher of the potential success of stable transformation. For this purpose, we developed a transient system to test the functionality of endosperm-preferred promoters in maize. Two endosperm-preferred promoters from rice (a globulin and a glutelin promoter) were employed. Ears of Zea mays L. were harvested at 17 d after pollination, surface sterilized and the endosperm excised. Using Agrobacterium tumefaciens co-cultivation and sonication, transient expression of the target genes was detected after 4 and 5 d. We demonstrate expression of CBH I and CHB II (both exo-cellulases) up to 1.7% TSP, under the rice globulin and glutelin promoters.     

Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition.

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.     

Manipulating corn germplasm to increase recombinant protein accumulation

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.