Carmen Menéndez

Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4

The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) (IsdA) was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the IsdA gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the A. diazotrophicus levansucrase with those of the Bacillus subtilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthetized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of A. diazotrophicus.

Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.

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.

Structural levansucrase gene (lsdA) constitutes a functional locus conserved in the species Gluconacetobacter diazotrophicus

Abstract. Levansucrase (EC 2.4.1.10) was identified as a constitutive exoenzyme in 14 Gluconacetobacter diazotrophicus strains recovered from different host plants in diverse geographical regions. The enzyme, consisting of a single 60-kDa polypeptide, hydrolysed sucrose to synthesise oligofructans and levan. Sugar-cane-associated strains of the most abundant genotype (electrophoretic type 1) showed maximal values of levansucrase production. These values were three-fold higher than those of the isolates recovered from coffee plants. Restriction fragment length polymorphism analysis revealed a high degree of conservation of the levansucrase locus (lsdA) among the 14 strains under study, which represented 11 different G. diazotrophicus genotypes. Targeted disruption of the lsdA gene in four representative strains abolished their ability to grow on sucrose, indicating that the endophytic species G. diazotrophicus utilises plant sucrose via levansucrase.

Molecular Cloning and Expression in Escherichia coli of an Exo-Levanase Gene from the Endophytic Bacterium Gluconacetobacter diazotrophicus SRT4

Gluconacetobacter diazotrophicus produces levan from sucrose by a secreted levansucrase (LsdA). A levanase-encoding gene (lsdB), starting 51 bp downstream of the lsdA gene, was cloned from strain SRT4. The lsdB gene (1605 bp) encodes a protein (calculated molecular mass 58.4 kDa) containing a putative 36-amino-acid signal peptide at the N-terminus. The deduced amino acid sequence shares 34%, 33%, 32%, and 29% identities with levanases from Actinomyces naeslundii, Bacillus subtilis, Paenibacillus polymyxa, and Bacteroides fragilis, respectively. The lsdB expression in Escherichia coli under the control of the T7 RNA polymerase promoter resulted in an active enzyme which hydrolyzed levan, inulin, 1-kestose, raffinose, and sucrose, but not melezitose. Levanase activity was maximal at pH 6.0 and 30°C, and it was not inhibited by the metal ion chelator EDTA or the denaturing agents dithiothreitol and β-mercaptoethanol. The recombinant LsdB showed a fourfold higher rate of hydrolysis on levan compared to inulin, and the reaction on both substrates resulted in the successive liberation of the terminal fructosyl residues without formation of intermediate oligofructans, indicating a non-specific exo-levanase activity.

Levansucrase from Acetobacter diazotrophicus SRT4 Is Secreted via Periplasm by a Signal-Peptide-Dependent Pathway

Abstract.Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay—matrix-assisted laser desorption/ionization—time-of-flight mass spectrometry). Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.

A thermostable exo-β-fructosidase immobilised through rational design

Thermotoga maritima exo-β-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimally immobilised on Glyoxyl-Sepharose CL 4B using the Rational Design of Immobilised Derivatives (RDID) strategy. Covalent attachment of the N-glycosylated BfrA onto the activated support at pH 10 allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variation in the catalytic properties of the enzyme and allowed further enhancement of the thermal stability. Complete inversion of cane sugar (2.04 M) in a batch stirred tank reactor at 60 °C was achieved with a productivity of 22.2 g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilised enzyme of 5 days at 60 °C was determined in a continuously operated fixed-bed column reactor. Our results promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.

Transcriptional Regulation and Signal-Peptide-Dependent Secretion of Exolevanase (LsdB) in the Endophyte Gluconacetobacter diazotrophicus

ABSTRACT Gluconacetobacter diazotrophicus utilizes plant sucrose with a constitutively expressed levansucrase (LsdA), producing extracellular levan, which may be degraded under energetically unfavored conditions. Reverse transcriptase-PCR analysis revealed that lsdA and the downstream exolevanase gene (lsdB) form an operon. lsdB transcription was induced during growth with low fructose concentrations (0.44 to 33 mM) and repressed by glucose. Transport of LsdB to the periplasm involved N-terminal signal peptide cleavage. Type II secretion mutants failed to transfer LsdB across the outer membrane, impeding levan hydrolysis.

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 β(2,6)-linked fructosyl chains with β(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 β(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.

Kinetics of Sucrose Hydrolysis by Immobilized Recombinant Pichia pastoris Cells in a Batch reactors

Sucrose hydrolysis was carried out in a constant-volume batch reactor, using recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase, entrapped in calcium alginate beads. The kinetics of the enzymatic hydrolysis of sucrose by the biocatalyst was examined at substrate concentrations ranging between 0.03 M and 2.04 M. The reaction rate increases until 0.31 M after which the reaction velocity was constant until 1.16 M, above this concentration, the reaction rate decreases with increasing sucrose concentration. The experimental data obtained with two weight of the biocatalyst were incorporated into two kinetic models to predict the reaction time needed for sucrose hydrolysis. One model was applied for sucrose concentrations bellow 1.16 M while a second one could be used at inhibitory range between 1.46 and 2.04 M with a k value as function of initial sucrose concentration and biocatalyst weight.