Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase.

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate.

BACKGROUND: An ideal immobilized biocatalyst for the industrial-scale production of invert sugar should stably operate at elevated temperatures (60-70°C) and high sucrose concentrations (above 60%, w/v). Commercial invertase from the yeast Saccharomyces cerevisiae is thermolabile and suffers from substrate inhibition. Thermotoga maritima ß-fructosidase (BfrA) is the most thermoactive and thermostable sucrose-hydrolysing enzyme so far identified and allows complete inversion of the substrate in highly concentrated solutions. RESULTS: In this study, heat-killed Pichia pastoris cells bearing N-glycosylated BfrA in the periplasmic space were entrapped in calcium alginate beads. The immobilized recombinant yeast showed maximal sucrose hydrolysis at pH 5-7 and 90°C. BfrA was 65% active at 60°C and had no activity loss after incubation without the substrate at this temperature for 15 h. Complete inversion of cane sugar (2.04 M) at 60°C was achieved in batchwise and continuous operation with respective productivities of 4.37 and 0.88 gram of substrate hydrolysed per gram of dry beads per hour. The half-life values of the biocatalyst were 14 and 20 days when operated at 60°C in the stirred tank and the fixed-bed column, respectively. The reaction with non-viable cells prevented the occurrence of sucrose fermentation and the formation of by-products. Six-month storage of the biocatalyst in 1.46 M sucrose (pH 5.5) at 4°C caused no reduction of the invertase activity. CONCLUSIONS: The features of the novel thermostable biocatalyst developed in this study are more attractive than those of immobilized S. cerevisiae cells for application in the enzymatic manufacture of inverted sugar syrup in batch and fixed-bed reactors.

Constitutive high-level expression of a codon-optimized ß-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable ß-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.

Microbial signature profiles of Penaeus vannamei larvae in low-survival hatchery tanks affected by vibriosis.

Vibriosis is caused by some pathogenic Vibrio and produces significant mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei larvae in commercial hatcheries. Acute hepatopancreatic necrosis disease (AHPND) is an emerging vibriosis affecting shrimp-producing countries worldwide. Zoea 2 syndrome is another type of vibriosis that affects the early stages of P. vannamei larvae. Although the pathogenesis of AHPND and zoea 2 syndrome is well known, there is scarce information about microbial composition and biomarkers of P.vannamei larvae affected by AHPND, and there is no study of the microbiome of larvae affected by zoea 2 syndrome. In this work, we characterized the microbiome of P. vannamei larvae collected from 12 commercial hatchery tanks by high-throughput sequencing. Seven tanks were affected by AHPND, and five tanks were affected by zoea 2 syndrome. Subsequently, all samples were selected for sequencing of the V3-V4 region of the16S rRNA gene. Similarity analysis using the beta diversity index revealed significant differences in the larval bacterial communities between disease conditions, particularly when Vibrio was analyzed. Linear discriminant analysis with effect size determined specific microbial signatures for AHPND and zoea 2 syndrome. Sneathiella, Cyclobacterium, Haliea, Lewinella, among other genera, were abundant in AHPND-affected larvae. Meanwhile, Vibrio, Spongiimonas, Meridianimaribacter, Tenacibaculum, among other genera, were significantly abundant in larvae affected by zoea 2 syndrome. The bacterial network at the phylum level for larvae collected from tanks affected by AHPND showed greater complexity and connectivity than in samples collected from tanks affected by zoea 2 syndrome. The bacterial connections inter Vibrio genera were higher in larvae from tanks affected by zoea 2 syndrome, also presenting other connections between the genera Vibrio and Catenococcus. The identification of specific biomarkers found in this study could be useful for understanding the microbial dynamics during different types of vibriosis.

Microbiome of Penaeus vannamei Larvae and Potential Biomarkers Associated With High and Low Survival in Shrimp Hatchery Tanks Affected by Acute Hepatopancreatic Necrosis Disease.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging bacterial disease of cultured shrimp caused mainly by Vibrio parahaemolyticus, which harbors the lethal PirAB toxin genes. Although Penaeus vannamei (P. vannamei) postlarvae are susceptible to AHPND, the changes in the bacterial communities through the larval stages affected by the disease are unknown. We characterized, through high-throughput sequencing, the microbiome of P. vannamei larvae infected with AHPND-causing bacteria through the larval stages and compared the microbiome of larvae collected from high- and low-survival tanks. A total of 64 tanks from a commercial hatchery were sampled at mysis 3, postlarvae 4, postlarvae 7, and postlarvae 10 stages. PirAB toxin genes were detected by PCR and confirmed by histopathology analysis in 58 tanks. Seven from the 58 AHPND-positive tanks exhibited a survival rate higher than 60% at harvest, despite the AHPND affectation, being selected for further analysis, whereas 51 tanks exhibited survival rates lower than 60%. A random sample of 7 out of these 51 AHPND-positive tanks was also selected. Samples collected from the selected tanks were processed for the microbiome analysis. The V3-V4 hypervariable regions of the 16S ribosomal RNA (rRNA) gene of the samples collected from both the groups were sequenced. The Shannon diversity index was significantly lower at the low-survival tanks. The microbiomes were significantly different between high- and low-survival tanks at M3, PL4, PL7, but not at PL10. Differential abundance analysis determined that biomarkers associated with high and low survival in shrimp hatchery tanks affected with AHPND. The genera Bacillus, Vibrio, Yangia, Roseobacter, Tenacibaculum, Bdellovibrio, Mameliella, and Cognatishimia, among others, were enriched in the high-survival tanks. On the other hand, Gilvibacter, Marinibacterium, Spongiimonas, Catenococcus, and Sneathiella, among others, were enriched in the low-survival tanks. The results can be used to develop applications to prevent losses in shrimp hatchery tanks affected by AHPND.

Fructooligosaccharides production by Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris.

The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Tall fescue (Schedonorus arundinaceus). The increase of the transgene dosage from one to nine copies enhanced 7.9-fold the recombinant enzyme (Sa1-SSTrec) yield without causing cell toxicity. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (38%) and extracellular release (62%) of Sa1-SSTrec to an overall activity of 102.1 U/ml when biomass reached (106 g/l, dry weight) in fed-batch fermentation using cane sugar for cell growth. The volumetric productivity of the nine-copy clone PGFT6x-308 at the end of fermentation (72 h) was 1422.2 U/l/h. Sa1-SSTrec purified from the culture supernatant was a monomeric glycoprotein optimally active at pH 5.0-6.0 and 45-50 °C. The removal of N-linked oligosaccharides by Endo Hf treatment decreased the enzyme stability but had no effect on the substrate and product specificities. Sa1-SSTrec converted sucrose (600 g/l) into 1-kestose (GF2) and nystose (GF3) in a ratio 9:1 with their sum representing 55-60% (w/w) of the total carbohydrates in the reaction mixture. Variations in the sucrose (100-800 g/l) or enzyme (1.5-15 units per gram of substrate) concentrations kept unaltered the product profile. Sa1-SSTrec is an attractive candidate enzyme for the industrial production of short-chain fructooligosaccharides, most particularly 1-kestose.

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.

High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene.

Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long ß(2,6)-linked fructosyl chains with ß(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded ß(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.

A type II protein secretory pathway required for levansucrase secretion by Gluconacetobacter diazotrophicus.

The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys(162) by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

Propuesta metodológica para la determinación de las épocas climáticas con aplicación a los estudios de producción y reproducción bovina en el trópico / Methodological proposal to determine the climate periods applied to production and reproduction studies on bovine cattle in the tropics

El efecto del clima sobre el comportamiento productivo y reproductivo en plantas y animales se evidencia a través de la producción de forraje de la pradera, la ganancia de peso de los animales o la distribución de sus partos a lo largo del año. Con el fin de determinar el período climático más favorable para el desempeño productivo del ganado es prioritario caracterizar y clasificar el clima con base en losfactores que más lo afectan. La caracterización se logra a través de la elaboración del balance hídrico o contabilidad del agua en el suelo, metodología que tiene en cuenta la precipitación pluvial, la evapotranspiración potencial y real y el movimiento del agua a través del perfil del suelo. El efecto del clima sobre los parámetros productivos y reproductivos se evalúa tomando en cuenta las características climáticas en los cuatro o cinco meses previos a la fecha de corte del análisis y con base en este criterio se crea la base de datos. El análisis estadístico emplea los procedimientos del Modelo Lineal General (GLM) del Statistical Analysis System (SAS) y los coeficientes de correlación y regresión. La metodología climática y estadística propuesta es aplicable a los sistemas de producción animal como una herramienta para el análisis e interpretación de los eventos productivos y reproductivos en los bovinos.