Global and grain-specific accumulation of glycoside hydrolase family 10 xylanases in transgenic maize (Zea mays).

In planta expression of cell wall degrading enzymes is a promising approach for developing optimized biomass feedstocks that enable low-cost cellulosic biofuels production. Transgenic plants could serve as either an enzyme source for the hydrolysis of pretreated biomass or as the primary biomass feedstock in an autohydrolysis process. In this study, two xylanase genes, Bacillus sp. NG-27 bsx and Clostridium stercorarium xynB, were expressed in maize (Zea mays) under the control of two different promoters. Severe phenotypic effects were associated with xylanase accumulation in maize, including stunted plants and sterile grains. Global expression of these xylanases from the rice ubiquitin 3 promoter (rubi3) resulted in enzyme accumulation of approximately 0.01 mg enzyme per gram dry weight, or approximately 0.1% of total soluble protein (TSP). Grain-specific expression of these enzymes from the rice glutelin 4 promoter (GluB-4) resulted in higher-level accumulation of active enzyme, with BSX and XynB accumulating up to 4.0% TSP and 16.4% TSP, respectively, in shriveled grains from selected T0 plants. These results demonstrate the potential utility of the GluB-4 promoter for biotechnological applications. The phenotypic effects of xylanase expression in maize presented here demonstrate the difficulties of hemicellulase expression in an important crop for cellulosic biofuels production. Potential alternate approaches to achieve xylanase accumulation in planta without the accompanying negative phenotypes are discussed.

Improved performance of Eimeria-infected chickens fed corn expressing a single-domain antibody against interleukin-10.

Antimicrobial resistance is a significant challenge for human and animal health, and developing effective antibiotic-free treatments is a strategy to help mitigate microbial resistance. The global poultry industry faces growing challenges from Eimeria-induced coccidiosis, a serious enteric disease of chickens that currently requires treatment using ionophore antibiotics. Eimeria stimulates interleukin-10 (IL-10) expression in the small intestine and caecum of infected chickens, suppressing their immune response and facilitating disease progression. Single-domain antibodies raised from llamas immunized with chicken IL-10 (cIL-10) were developed that bind cIL-10 in vitro, block cIL-10 receptor binding and induce interferon gamma (IFN-γ) secretion from cIL-10-repressed primary chicken splenocytes. Single-domain antibodies expressed in transgenic corn demonstrated significant accumulation in phenotypically normal plants. When fed to Eimeria-challenged chickens, the transgenic corn significantly improved body weight gain (equal to that of salinomycin-treated animals), normalized the feed conversion ratio (to the same level as uninfected control animals), lowered E. tenella lesion scores to those of salinomycin-treated control animals, and reduced oocyst counts below those of infected untreated control animals. Here, we propose that transgenic corn may have a role in reducing the use of antibiotics in poultry production and maintaining animal health and productivity, and may contribute to efforts against global antimicrobial resistance.

Characterization and plant expression of a glyphosate-tolerant enolpyruvylshikimate phosphate synthase.

BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials. CONCLUSION: These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.

Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing.

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.