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.

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.

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 Cys162 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(AU)

El endofíticos diazotroph Gluconacetobacter diazotrophicus segrega un constitutivamente expresado levansucrase (LSDA, CE 2.4.1.10) para utilizar la planta de sacarosa. LSDA, a diferencia de otros levansucrases extracelular de bacterias gram-negativas, es transportado a la periplasm por un péptido señal dependiente de la vía. Se identificaron un grupo muy organizado de genes de codificación, al menos, los componentes LsdG,-O,-E,-F,-H, I, J, L-,-H,-N, y-D de un sistema de tipo II secretoras LSDA necesario para la translocación a través de la membrana externa. Otro marco de lectura abierta, designada lsdX, está situado entre el promotor y el operón lsdG, pero no fue identificado en las búsquedas BLASTX de la DDBJ / EMBL / bases de datos GenBank. El lsdX,-G, y O-genes fueron aislados de una cepa de la biblioteca cósmido SRT4 por la complementación de un mutante etílico methanesulfonate LSDA incapaz de transporte a través de la membrana externa. Los genes lsdE abajo,-F,-H, I, J, L-,-H,-N, y-D cromosómicas fueron aislados a través de a pie. El alto contenido de G + C (64 a 74%) y el codón de uso de los genes identificados son coherentes con el contenido de G + C y el codón de uso de la norma G. diazotrophicus gen estructural. Análisis de secuencias de genes de la agrupación indicó que una transcripción polycistronic se sintetiza. Dirigida interrupción de lsdG, lsdO, bloqueados o lsdF LSDA secreción, y la bacteria no crezca en sacarosa......

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.