Levansucrase Secretion Research Articles

Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements.

Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.

Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His(6)- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His(6)-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates D-Gal-Fru, D-Xyl-Fru, D-Man-Fru, and D-Fuc-Fru.

The effect of amphiphilic compounds on the secretion of levansucrase by Zymomonas mobilis

The effect of some aliphatic (n-butanol to n-hexadecanol) and aromatic (benzyl and phenethyl alcohols), anesthetics (procaine) and surfactants (Tween 20 to Tween 80) on the secretion of levansucrase by the levan-producing strain of Gram-negative ethanologenic bacteria Zymomonas mobilis 113S were examined in this study.During incubation of Z. mobilis cells with sucrose (10mM) a decrease of the levansucrase activity was observed in the presence of these amphiphilic compounds concomitantly with an increase of a total amount of protein in the medium. Since none of the compounds under study had any effect on enzyme activity in vitro observed structure- and concentration-dependent relationships most probably reflected differently conditioned processes of membrane-associated secretion of levansucrase and total protein by Z. mobilis. The patterns of fluorescence titrations by ANS− indicated to competitive interactions between an amphiphilic compound of varied structure and the probe for the polar and non-polar binding sites of Z. mobilis membrane structures. The effect of 2,4-DNP (protonophore) and sodium azide (an inhibitor of ATPase) alone as well as in combination with aliphatic alcohols suggested to the participation of energy transduction system in the secretion of levansucrase by Z. mobilis cells. Under conditions of abolished proton motive force (PMF) the level of levansucrase decreased whereas the amount of protein elevated significantly in the medium in accordance with the expected requirement of PMF to perform the secretion of levansucrase and to keep intact the permeability barrier of cells.

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.

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8–1%, in which approximately 20–50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.

Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis.

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and alpha-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, alpha-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the alpha-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.

Extracellular secretion of levansucrase from

Extracellular secretion of levansucrase from

Bacillus subtilis levansucrase: the efficiency of the second stage of secretion is modulated by external effectors assisting folding

We investigated whether the concentration of H+ or metal ions such as Ca2+, or both, on the external side of the cytoplasmic membrane is involved in coupling of folding and secretion of Bacillus subtilis levansucrase by studying the modulation of each isolated event. In vitro at 30°C, in the absence of Ca2+, the equilibrium between the unfolded and the folded states of levansucrase was rapidly and totally displaced toward the folded state by a small pH shift from 7·4 to 6·0. Ca2+ (> 5 mM) acted as a catalyst of folding at pH ≥ 7. In vivo pulse-chase experiments at 30°C showed that, in the absence of Ca2+, the rate and the yield of the second step of levansucrase secretion were strongly dependent on the external pH. In acidic growth medium (pH 5·8), secretion was efficient. In contrast, at pH ≥ 7, the presence of Ca2+ was essential for secretion. In bacteria grown at high temperature (48°C), both external acidic pH and Ca2+ were required for efficient secretion. Moreover, a levansucrase variant altered in its calcium affinity was efficiently secreted only under acidic growth conditions. Depending on the culture conditions, the differences in H+ or Ca2+ concentrations which are maintained between the opposite sides of the energized cytoplasmic membrane could be adequate to catalyse conformational transition which could play a critical role in the second step of the protein release. These environmental parameters could also affect the yield of secretion of some other secretory proteins, leading to the hypothesis that several different secretion mechanisms could coexist in B. subtilis.

Levan and levansucrase synthesized by the fireblight pathogen Erwinia amylovora

The fireblight pathogen Erwinia amylovora synthesizes two different types of extracellular polysaccharide (EPS) when cultured in vitro, an acidic and a neutral compound. Neutral EPS was prepared from culture supernatants by cross-flow ultrafiltration and molecular washing, and purified by column chromatography. This material represented most of the crude EPS, which was produced in high-sucrose medium. It was entirely composed of fructose. The signals obtained from the EPS-preparation by 13C-NMR spectroscopy were identical with those of levan from Aerobacter levanicum indicating a β-2,6 linkage of the sugar residues. The neutral component of the EPS was thus identified as levan (β-2,6- d-fructofuranan). In the protein fraction associated with crude EPS, the levan polymerizing enzyme levansucrase (β-2,6- d-fructan: d-glucose 6-fructosyltransferase, EC 2.4.1.10) was detected. The enzyme was also produced at similar levels with several carbon sources by the bacterium. In polyacrylamide gel eletrophoresis its molecular weight under completely denaturating conditions was 46 kDa, when purified to apparent homogeneity by membrane-exchange chromatography. However, in isoelectric focussing, the enzyme preparation produced two bands at pI 4·0 and pI 3·6, respectively. Both bands displayed enzyme activity in an in situ levansucrase assay. Secretion of levansucrase may play an important role for multiplication of E. amylovora in sucrose containing plant tissue.

Levansucrase: a tool to study protein secretion in Bacillus subtilis

The Bacillus amyloliquefaciens levansucrase gene ( sacB[BamP]) was engineered in such a way that a heterologous gene could be inserted between the second and third codon of the mature levansucrase. Extracellular levansucrase activity was detected only when the heterologous protein was secreted into the growth medium. A positive selection system to isolate suppressors of signal sequence mutants in Bacillus subtilis has been developed based on the secretion of levansucrase.

Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis

A series of alterations in the Bacillus amyloliquefaciens levansucrase signal peptide were made by in vitro mutagenesis, and their effect on the secretion of levansucrase in Bacillus subtilis was studied. Some of the alterations resulted in a completely defective signal peptide. These included the removal of positively charged residues from the N-terminus and disruption of the hydrophobic core of the signal peptide either by introducing a charged residue or by deleting five or more amino acids. Analysis of the signal peptide processing-site alterations revealed that small residues are preferred at the -1 and -3 positions. However, a wide variety of amino acids are tolerated at the +1 position.

Expression of levansucrase- -galactosidase hybrids inhibits secretion and is lethal in Bacillus subtilis

The lacZ gene of Escherichia coli was fused to several positions downstream from the 5' end of the Bacillus subtilis sacB gene, which encodes levansucrase (LS), a sucrose-inducible extracellular enzyme. Effects of hybrid protein expression in B. subtilis were studied. Several fusions were tested, and two significantly interfered with growth of cells and with LS secretion when induced with sucrose. Chromosomal amplification of the fusions, leading to strong expression of the hybrid proteins, completely blocked LS secretion and was lethal for B. subtilis when expression was induced.

Levansucrase of Bacillus subtilis: effects on the secretion process of single amino acid substitutions in the mature part of the protein.

Substitutions of an aspartate or an arginine residue for the glycine residue at position 366 of the mature part of Bacillus subtilis levansucrase were obtained by mutagenesis. Quantitative estimation from immunoblot analysis showed that the two transient membrane forms of the modified proteins were present in the membrane at the same level as that of the wild-type protein. The proteolytic processing, which was previously shown to be the first step of the levansucrase secretion process, was not affected in these modified proteins. Results from pulse-chase experiments showed that the half-times for secretion of the modified levansucrases into the culture medium were nearly the same as that of the wild-type protein, but the amount of the modified proteins secreted was significantly reduced. Purified samples of the modified enzymes were subtilisin insensitive and possessed enzyme activities very similar to those of the wild-type enzyme. The results suggest that the 366 site probably belongs to a functional domain of the protein which could play an important role in the second step of the levansucrase secretion process.

Secretion Mechanism of Bacillus subtilis Levansucrase: Characterization of the Second Step

The kinetics of levansucrase secretion were examined in a strain of Bacillus subtilis which overproduces the enzyme (sacUh). Pulse-labelling experiments indicated that the second step of the levansucrase secretion process has the properties of a membrane active-transport system. This event appears to be directly linked to the influx of iron into the bacteria. The response of B. subtilis to the inducer of levansucrase synthesis was modulated by ferrichelators and the extent of the response varied with the nature of the ferrichelator. Ferridihydroxybenzoate markedly shortened the induction lag period. It is inferred from these data that iron occurs as a cofactor for components of the membrane sites of synthesis/secretion of B. subtilis levansucrase.

5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis.

The regulation of the levansucrase gene sacB was studied in Bacillus subtilis strains. Fusions were constructed in which genes of cytoplasmic proteins such as lacZ were placed immediately downstream from sacR, the regulatory region located upstream from sacB. These fusions were introduced in mutants affected in sacB regulation. In all cases the marker gene was affected in the same way as sacB by the genetic context. This result is of particular interest for the sacU pleiotropic mutations, which affect sacB expression and other cellular functions such as the synthesis of several exocellular enzymes. We also showed that strains harboring sacU+ or sacU-hyperproducing alleles contained different amounts of sacB mRNA, which was proportional to their levansucrase secretion. We concluded that the sacU gene does not affect sacB expression at the level of secretion but acts on a target within sacR. We discuss the possibility that sacU acts on a part of sacR, a homologous copy of which was found upstream from the gene of another sacU-dependent secreted enzyme of B. subtilis, beta-glucanase.