Levansucrase Activity Research Articles

CLONING AND EXPRESSION OF LEVANSUCRASE (SacB) GENE FROM BACILLUS LICHENIFORMIS MJ8 IN ESCHERICHIA COLI AND ENZYMATIC SYNTHESIS OF LEVAN

In this study, a highly levansucrase-producing strain was isolated, identified as Bacillus lichniformans MJ8, and registered with the accession number OM672244.1 in the NCBI database. The SacB gene responsible for levansucrase production was transferred from this bacterium into Escherichia coli. It was found that the gene contains 1449 bp nucleotides, encoding 482 amino acids. The gene has been given the accession number ON811641.1 in the NCBI gene bank. The transformation was achieved by a cloning the SacB gene to plasmid pTG19-T, which was transferred to Escherichia coli DH5α. The Escherichia coli BL21 (DE3), with pet-28a (+) vector, was used to express the gene. One mM IPTG is induces the cloned gene to produce SacB protein. The levansucrase activity was 14.31 U/ml after transformation. The study also included the identification and characterization of levan produced by the bacteria using HPLC and FTIR techniques.

Purification and Characterization of Recombinant Levansucrase From Bacillus lichniformans MJ8

The ability to produce levansucrase was estimated in fifty Bacillus spp. isolates obtained from various sources. One isolate was found to be the higher producer of this enzyme through primary and secondary screening, which was performed by estimating levansucrase activity. It was found to be 6.05 U/ml. The results show, after identification by the Vitek 2 compact system with 92% probability and 16S ribosomal RNA gene sequencing, that this isolate is Bacillus licheniformis, designated in this study as MJ8, and registered in the Gene Bank under the accession number OM672244.1. Then, the gene of (SacB) was studied in detail. It contained 1449 nucleotides (accession number ON811641.1), expressed for 482 amino acids and 29 amino-acid as signal peptides. A recombinant plasmid, pET28a(+), with the levansucrase (SacB) gene, was over expressed in high efficiency E. coli BL21 (DE3). Then the enzyme was purified in three steps; firstly, it was precipitated with ammonium sulfate 40-80%. Secondly, DEAE cellulose ion exchange chromatography was applied then the gel filtration chromatography. The activity of levansucrase at the final purification steps reached 48.65 U/ml with a purification fold of 18.0 and a recovery of 48.9%. The levansucrase characterization was also studied. It was found that the molecular weight by SDS-PAGE and gel filtration methods was 43 kDa and 47 kDa, respectively, and the optimum temperature activity and stability were 45 ◦C, 30-35 ◦C, respectively while the pH activity and stability were 6.0, and 7.0, The levan was produced from cloned (SacB) levansucrase, separated, purified, and identified by NMR, AFM, and SEM analyses showed that its structure mostly consists of fructose residues.

Optimization strategy of Bacillus subtilis MT453867 levansucrase and evaluation of levan role in pancreatic cancer treatment

Pancreatic cancer is the fourth most lethal cancer type worldwide. Due to multiple levan applications including anticancer activities, studies related to levansucrase production are of interest. To our knowledge, levan effect on pancreatic cancer cells has not been tested previously. In this work, among eighteen bacterial honey isolates, Bacillus subtilis MT453867 showed the highest levan yield (33 g/L) and levansucrase production (8.31 U/mL). One-factor-at-a-time technique increased levansucrase activity by 60% when MgSO4 was eliminated. The addition of 60 g/L banana peels enhanced the enzyme activity (192 U/mL). Placket Burman design determined the media composition for maximum levan yield (54.8 g/L) and levansucrase production (505 U/mL). The identification of levan was confirmed by thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C-nuclear-magnetic resonance, and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. The LD50 of crude levan was 4833 mg/kg body weight. Levan had strong antioxidant activity and significantly reduced the expression of CXCR4 and MCM7 genes in pancreatic cancer cells with significant DNA fragmentation. In conclusion, Bacillus subtilis MT453867 levan is a promising adjunct to pancreatic-anticancer agents with both anti-cancer and chemoprotective effects.

Production and prebiotic properties of oligofructans from sugarcane juice fermentation by Bacillus subtilis TISTR 001.

Oligofructans are potential biological substances that due to their distinctive properties have a positive health-promoting effect. This research aimed to produce oligofructans using sugarcane juice fermentation with Bacillus subtilis TISTR 001 and to study the prebiotic properties in vitro. The results showed that the maximum total oligofructans in the form of free fructose was 2.57% (w/v) at the 84th hour of fermentation with 0.17g/g reducing sugar and a production yield of 0.031g/L/h and maximum levansucrase activity of 1.57 × 106U/mL at that time. The oligofructans contained in the fermented juice had potential functional ingredients that exhibited prebiotic properties that could resist the digestion of enzymes in the gastrointestinal tract under in vitro conditions with digestion of only 6.92%. In addition, the fermented juice promoted the growth of prebiotics, especially Bifidobacterium bifidum TISTR 2129 and inhibited the growth of pathogens using both single culturing and co-culturing with probiotics.

Levan from Leuconostoc citreum BD1707: production optimization and changes in molecular weight distribution during cultivation

BackgroundLevan is a well-known homopolymer of fructose composed predominantly of β-(2, 6) fructofuranosyl linkages in the backbone with occasional β-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated.MethodsThe cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).ResultsThe levan yield of BD1707 reached 34.86 g/L with a corresponding productivity of 7.47 g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26 °C and 200 rpm for 112 h in tomato juice supplemented with 172 g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320 × 107 Da at 6 h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809 × 106 Da after 120 h of cultivation.ConclusionThe levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected.

Recombinant Expression and Biochemical Characterization of Levansucrase from Halophilic Bacteria <i>Bacillus licheniformis</i> BK1 and BK2

Levansucrase was an extracellular polysacharride (EPS) which has a role in synthesizing levans by transferring fructose moiety from sucrose to acceptor molecules. In the previous study, we have successfully cloned the levansucarese gene from two Bacillus licheniformis strains of BK1 and BK2 labeled as lsbl-bk1 and lsbl-bk2. The present study aims to optimize the expression level of both genes in E. coli expression system and also to obtain the optimum conditions for the recombinant enzymes activity by applying the response surface methodology (RSM). The optimization result found that the highest Lsbl-bk1 production in E. coli expression system was occurred when the recombinant cells grown in the medium containing 0.6% (w/v) NaCl at 42°C, and induced by 0.6 mM IPTG. Different optimum conditions were found for Lsbl-bk2 production. It was achieved when 1.1% (w/v) NaCl added to the production medium and induced by 0.7 mM IPTG at 40°C. RSM optimization result for biochemical characterization of Lsbl-bk1 levansucrase showed the highest specific activity achieved at 56°C and pH 7.5, whereas for the Lsbl-bk2 levansucrase reached the highest specific activity at 50°C and pH 7.5. The addition of Co2+, Ti2+, Mg2+, Ba2+, Zn2+, Fe3+, Ca2+ metal ion to both levansucrases solution did not significantly altered their specific activity, indicating that both levansucrases are not metalo enzymes. Furthermore, the specific activity of levansucrase was also not affected by the addition of 1-25% (w/v) NaCl, suggesting that the variation of ionic strength did not alter the native state of both enzymes. The plot results of levansucrase specific activities toward sucrose concentration showed that both levansucrases follow Michaelis-Menten profile with kcat/KM values ​​about 3.8 and 3.6 s-1/mM respectively. These data indicated that the recombinant levansucrases from halophilic bacteria B. licheniformis BK1 and BK2 are a non metaloenzyme with high affinity and binding rate to sucrose substrate, in which the catalytic efficiency on hydrolysis reactions is relatively low.

Orchestration of virulence factor expression and modulation of biofilm dispersal in Erwinia amylovora through activation of the Hfq-dependent small RNA RprA.

Erwinia amylovora is the causative agent of the devastating disease fire blight of pome fruit trees. After infection of host plant leaves at apple shoot tips, E. amylovora cells form biofilms in xylem vessels, restrict water flow, and cause wilting symptoms. Although E. amylovora is well known to be able to cause systemic infection, how biofilm cells of E. amylovora transit from the sessile mode of growth in xylem to the planktonic mode of growth in cortical parenchyma remains unknown. Increasing evidence has suggested the important modulatory roles of Hfq‐dependent small RNAs (sRNAs) in the pathogenesis of E. amylovora. Here, we demonstrate that the sRNA RprA acts as a positive regulator of amylovoran exopolysaccharide production, the type III secretion system (T3SS), and flagellar‐dependent motility, and as a negative regulator of levansucrase activity and cellulose production. We also show that RprA affects the promoter activity of multiple virulence factor genes and regulates hrpS, a critical T3SS regulator, at the posttranscriptional level. We determined that rprA expression can be activated by the Rcs phosphorelay, and that expression is active during T3SS‐mediated host infection in an immature pear fruit infection model. We further showed that overexpression of rprA activated the in vitro dispersal of E. amylovora cells from biofilms. Thus, our investigation of the varied role of RprA in affecting E. amylovora virulence provides important insights into the functions of this sRNA in biofilm control and systemic infection.

Evaluation of the antivirus activity of Enterococcus faecalis Esawy levan and its sulfated form

Abstract The screening of 15 honey isolates showed that the E. faecalis Esawy KR758759 was the most potent levansucrase producer. It recorded the highest levansucrase activity (50.55 U/mL) and the highest levan yield (37.50 g/L). The levan molecular weight was determined to be 85,658 Da. The yielded levan was classified into three forms as: crude levan (CL), dialyzed levan (DL) and sulfated levan (SL). The three levan forms were characterized by FTIR. The characterization of levan by FTIR revealed that the structure of levan was homologous to the standard levan sample. 13C and 1H NMR have successfully confirmed the levan structure. The antivirus activity of the three compounds was evaluated against the Newcastle disease virus (NDV). The CL recorded complete inhibition of NDV followed by DL (95%) and SL (90%). The three levan forms had an immunostimulant effect with variations degree. A mechanism was developed to suggest the role of levan as an anti-virus against NDV.

Insights into the pH-dependent, extracellular sucrose utilization and concomitant levan formation by Gluconobacter albidus TMW 2.1191

Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3–pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3–5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation.

Combination treatment of MCF-7 spheroids by Pseudomonas aeruginosa HI1 levan and cisplatin

Treatment of cancer with chemotherapy drugs is a challenge because of drug resistance and severe side effects. This study aimed to evaluate the cytotoxicity of levan as a natural biopolymer alone and in combination with cisplatin. Consequently, honey isolate NRCA, Pseudomonas aeruginosa, recorded promising levansucrase activity and levan yield. Crude levan (CL) was dialyzed to dialyze levan (DL). Crude levan showed cytotoxicity effect by 69.5%, 74% on MCF-7 at 500 and 1000 μg/ml, respectively while it has a safe effect on normal cells. Dialyzed levan increased cancer cell viability. Combination treatment of CL and cisplatin significantly reduced the IC50 of cisplatin (9.4 μM) to 5.8 μM and 3.8 μM when combined with Levan 500 μg/ml and 1000 μg/ml, respectively. Crude levan cytotoxicity is a dose-independent with IC50 447 μg/ml. Levan did not cause DNA fragmentation in treated cells. Accordingly, treatment design to treat spheroids with a combination of both CL and cisplatin at different time intervals was suggested. This design was found to increase the cytotoxicity against MCF-7 spheroids and reduce the cytotoxicity of cisplatin on spheroids of normal cell line. It can be concluded that combination treatment of cisplatin and levan might decrease the IC50 of cisplatin and overcome side effects problems.

A close look on the effect of polyethylene glycol on the levansucrase thermostability: a case study of Brenneria sp. levansucrase.

To increase the low residual activity of levansucrase during long-time processing, an enhancement of its weak thermostability is needed. Here, the effect of metal ions and polyethylene glycol (PEG) on the thermostability of levansucrase from Brenneria sp. EniD312 were studied and evaluated. The residual activity was determined and the protein structure was evaluated by circular dichroism spectrum, fluorescence intensity (FI), and surface hydrophobicity (S0 ). As a result of incubation with 10% (w/v) PEG 4000, the enzyme activity was increased by 1.24-fold. After incubation with 5% PEG 4000 for 6h, the residual activity at 35 and 45 °C was decreased to 55% and 60% of the initial activity, with an increase of 1.2- and 3.3-fold than the wild-type enzyme. Furthermore, the random coil content of enzyme was decreased from 53% of the wild-type enzyme to 33.9% of the PEG pre-incubated enzyme. Additionally, the FI was maximally increased and the S0 was decreased from 117 to 69. All of these results suggested that after incubation with PEG 4000, the secondary and tertiary structure of wild-type enzyme could be greatly maintained and then its thermostability could be increased. This study was the first report on the enhancement of levansucrase thermostability by PEG incubation and might be a good guideline to other researches on levansucrase. © 2019 Society of Chemical Industry.

Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation.

Bacterial levansucrases produce β(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30–200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol−1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol−1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme.

Possible correlation between levansucrase production and probiotic activity of Bacillus sp. isolated from honey and honey bee.

Five bacterial isolates from honey and bee gut were selected based on their high levansucrase activity and levan yield which were strongly positively correlated. All isolates showed good tolerance to temperature up to 70 °C, to NaCl up to 3M and to 0.1% H2O2. They maintained over 59 and 64% survival at pH 9.0 and 2.0 respectively, but showed varying tolerance to 0.1% bile salts and pancreatic enzymes. Most isolates were susceptible to widely used antibiotics, but demonstrated diverse antimicrobial activity. Non hemolytic isolates were identified on the basis of 16S rRNA sequencing as Bacillus subtilis HMNig-2 and B. subtilis MENO2 with 97% homology. They exhibited promising probiotic characteristics and achieved highest levansucrase activity of 94.1 and 81.5U/mL respectively. Both exhibited highest biofilm formation ability in static microtiter plate assay. Also, they achieved 34 and 26% adhesion respectively to Caco-2cells and had highest free radical scavenging activity of 30.8 and 26.2% respectively. The levans of the two isolates showed good antimicrobial activity against some pathogens and exhibited positive prebiotic effect (prebiotic index >1) with Lactobacillus casei and Lactobacillus reuteri. Results suggest a correlation between levansucrase production, levan yield and pre-probiotic activities of the studied strains.

Levansucrase production by Zymomonas mobilis : Optimization of process parameters and fructooligosaccharide production

This article investigates the production and optimization of levansucrase using synthetic medium by Zymomonas mobilis NRRL B-14023. Response surface methodology was used to investigate the effects of three different fermentation parameters on levansucrase enzyme production. Maximum levansucrase activity of 13.3 µmol glucose/min was obtained at the optimum levels of process variables (pH 4.90, initial sucrose concentration 159.01 g/L and fermentation temperature 30.3°C). Response surface methodology was found to be useful in optimizing and determining the interactions among process variables. The purity of levansucrase used in enzyme assays were analyzed by SDS-PAGE and a dominant protein band of size around 47 kDa was obtained. The sequence of the produced enzyme was determined by peptide mass fingerprinting. The presence of kestose in the reaction mixture showed that Z. mobilis levansucrase can produce fructooligosaccharides. This study fulfills the lack of mathematical and statistical approaches in optimizing the levansucrase production by Z. mobilis. Practical applications Levansucrases are responsible for the production of valuable fructans; fructooligosaccharides (FOS) and levan. Fructooligosaccharides are sugar polymers three times less sweet than sucrose and naturally present in some foods. FOS have important promising use in the food industry due to their properties such as solubility in water, stability during storage, low calorie value, and prebiotic effects. In this research, the production of microbial levansucrase was optimized for potential industrial applications by response surface methodology. Future studies will focus on conversion of intrinsic sucrose of foods to FOS using levansucrase.

Expression and Characterization of Levansucrase from Clostridium acetobutylicum.

The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287 bp and encodes 428 amino acid residues, which could convert 150 mmol/L sucrose to levan with the liberation of glucose. The optimum pH and temperature of this enzyme for levan formation were pH 6 and 60 °C, respectively. Levansucrase activity of Ca-SacB was completely abolished by 5 mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were calculated to be 64 mmol/L and 190 μmol/min/mg, respectively. Interestingly, Ca-SacB was found to have high product specificity, and no fructooligosaccharide was identified in the product, indicating that Ca-SacB may be valuable for industrial production of levan. In addition, Ca-SacB is the first characterized levansucrase isolated from an anaerobic bacterium, which should be valuable for exploring new enzyme resources and deepening the understanding of the catalytic mechanisms of levansucrases.

Bacillus amyloliquefaciens levansucrase-catalyzed the synthesis of fructooligosaccharides, oligolevan and levan in maple syrup-based reaction systems

Maple syrups with selected degree Brix (°Bx) (15, 30, 60) were investigated as reaction systems for levansucrase from Bacillus amyloliquefaciens. The enzymatic conversion of sucrose present in the maple syrup and the production of the transfructosylation products were assessed over a time course of 48h. At 30°C, the use of maple syrup 30°Bx led to the highest levansucrase activity (427.53μmol/mg protein/min), while maple syrup 66°Bx led to the highest converted sucrose concentration (1.53M). In maple syrup 30°Bx, oligolevans (10<DP≤30) were synthesized as major products (>80%). In maple syrup 66°Bx, the most abundant products were oligolevans at 30°C and levans (DP≥30) at 8°C. The acceptor specificity study revealed the ability of B. amyloliquefaciens levansucrase to synthesize a variety of hetero-fructooligosaccharides (FOSs) in maple syrups 15°Bx and 30°Bx enriched with various disaccharides, with lactose being the preferred fructosyl acceptor. The current study is the first to investigate maple-syrup-based reaction systems for the synthesis of FOSs/oligolevans/levans.

Purification and characterization of an intracellular levansucrase derived from Bacillus methylotrophicus SK 21.002.

An intracellular levansucrase from Bacillus methylotrophicus SK 21.002 was isolated, purified, and characterized. The final specific levansucrase activity was 135.40 U/mg protein with an 11.78-fold enrichment and a 9.28% recovery rate. The molecular weight of the enzyme was approximately 60,000 Da as evaluated by gel filtration and SDS-PAGE. Both the maximum transfructosylation and hydrolytic activities were observed at pH 6.5. The enzyme exhibited optimum transfructosylation activity at 40 °C, whereas the optimum temperature of hydrolytic activity was 45 °C. Cu(2+), Fe(2+), Zn(2+), and Ni(2+) inhibited both the transfructosylation and hydrolytic activities up to 100%, whereas Mn(2+) inhibited only hydrolytic activity. Ca(2+) and Mg(2+) stimulated both transfructosylation and hydrolytic activities. The chemical modifiers (n-bromosuccinimide and phenylmethanesulfonyl fluoride) strongly inhibited hydrolytic and transfructosylation activity of the levansucrase. The Km and Vmax values of the purified levansucrase were 117.2 mM and 33.23 μmol/mg·Min, respectively. When the fructose concentration was below 0.2 M, higher fructose concentrations promoted the transfructosylation and inhibited the hydrolytic activity.

The stimulatory effect of mannitol on levan biosynthesis: Lessons from metabolic systems analysis of Halomonas smyrnensis AAD6T

Halomonas smyrnensis AAD(T) is a halophilic, gram-negative bacterium that can efficiently produce levan from sucrose as carbon source via levansucrase activity. However, systems-based approaches are required to further enhance its metabolic performance for industrial application. As an important step toward this goal, the genome-scale metabolic network of Chromohalobacter salexigens DSM3043, which is considered a model organism for halophilic bacteria, has been reconstructed based on its genome annotation, physiological information, and biochemical information. In the present work, the genome-scale metabolic network of C. salexigens was recruited, and refined via integration of the available biochemical, physiological, and phenotypic features of H. smyrnensis AAD6(T) . The generic metabolic model, which comprises 1,393 metabolites and 1,108 reactions, was then systematically analyzed in silico using constraints-based simulations. To elucidate the relationship between levan biosynthesis and other metabolic processes, an enzyme-graph representation of the metabolic network and a graph decomposition technique were employed. Using the concept of control effective fluxes, significant links between several metabolic processes and levan biosynthesis were estimated. The major finding was the elucidation of the stimulatory effect of mannitol on levan biosynthesis, which was further verified experimentally via supplementation of mannitol to the fermentation medium. The optimal concentration of 30 g/L mannitol supplemented to the 50 g/L sucrose-based medium resulted in a twofold increase in levan production in parallel with increased sucrose hydrolysis rate, accumulated extracellular glucose, and decreased fructose uptake rate.

Levans production by Gluconacetobacter diazotrophicus

Fil: Molinari, Maria Laura. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - la Plata. Cent.de Inv En Fermentaciones Industriales (i); Argentina