Properties of corn-expressed carbohydrase AC1 in swine diets and its effects on apparent ileal digestibility, performance, hematology, and serum chemistry.

Carbohydrases are often incorporated into livestock feed as digestive aids to improve animal performance. AC1 is a thermostable carbohydrase with ß-1,4-glucanase, endo-cellulase, and cellobiohydrolase activity. AC1 has been expressed in corn, where it accumulates in the grain for easy inclusion in animal diets. Incorporating the enzyme in high-fiber diets (corn-soy supplemented with distiller's dry grains with solubles) that were fed to 5-week-old pigs led to a trend of decreasing viscosity of the digesta as the dose of the enzyme increased (P = 0.092). AC1 also tended to increase the apparent ileal digestibility (AID) of neutral detergent fiber (P = 0.076). When fed diets containing 2126 U/kg AC1, pigs experienced no adverse effects in terms of performance metrics (body weights, average daily gain, average daily feed intake and gain-to-feed ratio), hematology, blood chemistry or general health when compared to pigs fed a control diet that lacked AC1.

Determining the phosphorus release of GraINzyme phytase in diets for nursery pigs.

The objective of this study was to determine the available P (aP) release curve for a new phytase source, GraINzyme Phytase (Agrivida Inc., Woburn, MA), which is expressed in corn containing an engineered Escherichia coli phytase called Phy02. Plant-expressed phytases are created by inserting phytase-encoding genes into plants resulting in their ability to produce seeds with increased concentrations of phytase. A total of 360 pigs (Line 200 × 400, DNA, Columbus, NE, initially 9.9 ± 0.19 kg) were used in a 21-d growth study. Pigs were weaned at approximately 21 d of age, randomly allotted to pens based on initial body weight (BW) and fed common starter diets. From days 18 to 21 postweaning, all pigs were fed a diet containing 0.11% aP. On day 21 postweaning, considered day 0 of the study, pens were blocked by BW and randomly allotted to one of eight dietary treatments with five pigs per pen and nine pens per treatment. Dietary treatments were formulated to include increasing aP derived from either an inorganic P source (0.11%, 0.19%, or 0.27% from monocalcium P) or increasing phytase (150, 250, 500, 1,000, or 1,500 FTU/kg). Diets were corn-soybean meal-based and contained 1.24% standardized ileal digestible Lys. On day 21 of the trial, one pig per pen (weighing closest to the mean pen BW) was euthanized and the right fibula was collected to determine bone ash using the nondefatted processing method. Overall (days 0 to 21), pigs fed increasing aP from inorganic P or phytase had increased (linear, P < 0.002) average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed (G:F; quadratic, P < 0.05). Bone ash weight (g) and percentage bone ash increased (linear, P < 0.001), with increasing inorganic P or added phytase. Based on the composition of the diets used in this study, the release equations developed for GraINzyme for ADG, G:F, bone ash weight, and percentage bone ash are as follows: aP = (0.255 × FTU)/(1299.969 + FTU), aP = (0.233 × FTU)/(1236.428 + FTU), aP = (45999.949 × FTU)/(462529200 + FTU), and aP = (0.272 × FTU)/(2576.581 + FTU), respectively.

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.

Effects of feeding corn-expressed phytase on the live performance, bone characteristics, and phosphorus digestibility of nursery pigs.

A 41-d feeding trial was conducted to determine the efficacy of a corn-expressed phytase (GZ; GraINzyme, Agrivida Inc., Woburn, MA) on the live performance, bone characteristics, and P digestibility of nursery pigs fed a reduced P diet. Weaned piglets (21 ± 3 d; n = 324) were acclimated on a common diet for 7 d (phase 1) before randomization into 54 single-sex pens (5 gilt and 4 barrow pens per treatment) containing 6 pigs (6.6 ± 1.2 kg) per pen. Six treatments were fed: positive control (PC; 0.4% or 0.32% aP for phase 2 or 3 and 4, respectively), negative control (NC; 0.15% reduction in aP), and 500, 1,000, 2,000, or 4,000 FTU per kg phytase from GZ added to NC in a 3-phase feeding program. Pigs were weighed on day 1, 14, 28, and 41, and feed disappearance recorded per phase. Apparent total tract digestibility (ATTD) of P was determined by feeding chromic oxide marker (day 28 to 35) and collecting fecal samples on day 35. On day 41, 4 pigs per pen were euthanized and metacarpal bones were collected to evaluate bone breaking strength (BBS) and ash. Data were analyzed using PROC GLM of SAS (block, sex, and treatment). Treatment least squares means were separated and linear and quadratic treatment effects evaluated. Other than feed efficiency (G:F) and day 15 to 28 ADFI, the pigs fed PC were superior (P < 0.05) to NC-fed pigs in all other variables. Pigs fed ≥500 FTU per kg phytase had increased (P < 0.05) ADG and ADFI compared to NC pigs and equivalent (P > 0.05) ADG and ADFI as PC pigs from day 0 to 41. Feeding ≥500 FTU per kg phytase resulted in higher (P < 0.05) ATTD of P than both NC and PC pigs and higher (P < 0.05) BBS and bone ash weight than NC. Pigs fed 1,000 or 2,000 FTU per kg phytase had equivalent (P > 0.05) BBS and bone ash weight compared to pigs fed PC diets. Feeding 4,000 FTU per kg phytase resulted in higher (P < 0.05) day 1 to 41 ADG, ATTD of P, and bone ash weight compared to feeding ≤1,000 FTU per kg phytase or PC diets. There were linear (P < 0.05) increases in ADG, ADFI, ATTD of P, BBS, and bone ash characteristics as GZ inclusion increased. In conclusion, ≥500 FTU per kg phytase from GZ improved growth, ATTD of P, BBS, and bone ash when added to a reduced P diet and 4,000 FTU per kg phytase increased growth greater than the PC treatment.

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.

A predictive model of intein insertion site for use in the engineering of molecular switches.

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch.

Incorporating genome-scale tools for studying energy homeostasis.

Mammals have evolved complex regulatory systems that enable them to maintain energy homeostasis despite constant environmental challenges that limit the availability of energy inputs and their composition. Biological control relies upon intricate systems composed of multiple organs and specialized cell types that regulate energy up-take, storage, and expenditure. Because these systems simultaneously perform diverse functions and are highly integrated, they are extremely difficult to understand in terms of their individual component contributions to energy homeostasis. In order to provide improved treatments and clinical options, it is important to identify the principle genetic and molecular components, as well as the systemic features of regulation. To begin, many of these features can be discovered by integrating experimental technologies with advanced methods of analysis. This review focuses on the analysis of transcriptional data derived from microarrays and how it can complement other experimental techniques to study energy homeostasis.

Metabolic engineering.

Metabolic engineering is a powerful methodology aimed at intelligently designing new biological pathways, systems, and ultimately phenotypes through the use of recombinant DNA technology. Built largely on the theoretical and computational analysis of chemical systems, the field has evolved to incorporate a growing number of genome scale experimental tools. This combination of rigorous analysis and quantitative molecular biology methods has endowed metabolic engineering with an effective synergism that crosses traditional disciplinary bounds. As such, there are a growing number of applications for the effective employment of metabolic engineering, ranging from the initial industrial fermentation applications to more recent medical diagnosis applications. In this review we highlight many of the contributions metabolic engineering has provided through its history, as well as give an overview of new tools and applications that promise to have a large impact on the field's future.

Regulation of mouse hepatic genes in response to diet induced obesity, insulin resistance and fasting induced weight reduction.

BACKGROUND: Obesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrays and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO)-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions. RESULTS: Transcriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced, hepatic glucose output via phosphoenolpyruvate carboxykinase (PCK1), whose mRNA levels were also elevated. CONCLUSION: These findings demonstrate that hepatocytes respond to DIO and weight reduction by controlling gene transcription in a variety of important molecular pathways. Future studies that characterize the physiological significance of the identified genes in modulating energy homeostasis could provide a better understanding of the mechanisms linking DIO with insulin resistance.

Dynamics of gene silencing by RNA interference.

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.

Elucidation of gene interaction networks through time-lagged correlation analysis of transcriptional data.

The photosynthetic cyanobacterium Synechocystis sp. strain PCC 6803 uses a complex genetic program to control its physiological response to alternating light conditions. To study this regulatory program time-series experiments were conducted by exposing Synechocystis sp. to serial perturbations in light intensity. In each experiment whole-genome DNA microarrays were used to monitor gene transcription in 20-min intervals over 8- and 16-h periods. The data was analyzed using time-lagged correlation analysis, which identifies genetic interaction networks by constructing correlations between time-shifted transcription profiles with different levels of statistical confidence. These networks allow inference of putative cause-effect relationships among the organism's genes. Using light intensity as our initial input signal, we identified six groups of genes whose time-lagged profiles possessed significant correlation, or anti-correlation, with the light intensity. We expanded this network by using the average profile from each group of genes as a seed, and searching for other genes whose time-lagged profiles possessed significant correlation, or anti-correlation, with the group's average profile. The final network comprised 50 different groups containing 259 genes. Several of these gene groups possess known light-stimulated gene clusters, such as Synechocystis sp. photosystems I and II and carbon dioxide fixation pathways, while others represent novel findings in this work.

Metabolic and transcriptional patterns accompanying glutamine depletion and repletion in mouse hepatoma cells: a model for physiological regulatory networks.

An important objective in postgenomic biology is to link gene expression to function by developing physiological networks that include data from the genomic and functional levels. Here, we develop a model for the analysis of time-dependent changes in metabolites, fluxes, and gene expression in a hepatic model system. The experimental framework chosen was modulation of extracellular glutamine in confluent cultures of mouse Hepa1-6 cells. The importance of glutamine has been demonstrated previously in mammalian cell culture by precipitating metabolic shifts with glutamine depletion and repletion. Our protocol removed glutamine from the medium for 24 h and returned it for a second 24 h. Flux assays of glycolysis, the tricarboxylic acid (TCA) cycle, and lipogenesis were used at specified intervals. All of these fluxes declined in the absence of glutamine and were restored when glutamine was repleted. Isotopomer spectral analysis identified glucose and glutamine as equal sources of lipogenic carbon. Metabolite measurements of organic acids and amino acids indicated that most metabolites changed in parallel with the fluxes. Experiments with actinomycin D indicated that de novo mRNA synthesis was required for observed flux changes during the depletion/repletion of glutamine. Analysis of gene expression data from DNA microarrays revealed that many more genes were anticorrelated with the glycolytic flux and glutamine level than were correlated with these indicators. In conclusion, this model may be useful as a prototype physiological regulatory network where gene expression profiles are analyzed in concert with changes in cell function.