Clarita Olvera

Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di‐rhamnolipid biosynthesis

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso‐active glycolipids containing one (mono‐rhamnolipid) or two (di‐rhamnolipid) l‐rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono‐rhamnolipid from dTDP‐l‐rhamnose and β‐hydroxydecanoyl‐β‐hydroxydecanoate, whereas di‐rhamnolipid is produced from mono‐rhamnolipid and dTDP‐l‐rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di‐rhamnolipid (l‐rhamnose‐l‐rhamnose‐β‐hydroxydecanoyl‐β‐hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of l‐rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII‐2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di‐rhamnolipid, whereas mono‐rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1‐rhlA) was incapable of producing both mono‐ and di‐rhamnolipid. Complementation of mutant RTII‐2 with plasmid pRTII‐26 containing rhlC restored the level of di‐rhamnolipid production in the recombinant to a level similar to that of the wild‐type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A σ54‐type promoter for the PA1131–rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131–rhlC operon was dependent on the P. aeruginosa rhl quorum‐sensing system, and a well‐conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di‐rhamnolipid.

Monorhamnolipids and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) production using Escherichia coli as a heterologous host

Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-l-rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.

Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase.

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.

Production of functional oligosaccharides through limited acid hydrolysis of agave fructans

A controlled acid thermal hydrolysis process of fructans from agave (Agave tequilana Weber var. azul) was designed to produce a mixture of functional prebiotic fructooligosaccharides and sweetening power. Despite its highly branched structure, first-order kinetic behaviour with respect to substrate concentration was found with an activation energy of 95kJ/mol, similar to the value found for other linear fructans such as chicory inulin. Fructose equivalent (FE) an analogous parameter to dextrose equivalent (DE) used in the starch industry was introduced to characterise fructan hydrolysis; maximum oligosaccharide production was observed when fructose equivalent (FE) reached 27-48 with a structural profile analysed by high-performance anion-exchange chromatography HPAEC-PAD. After hydrolysis, glucose and fructose may be eliminated through a biological purification step involving the addition of Pichia pastoris cells, which selectively consume these sugars but are unable to metabolise fructooligosaccharides.

Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties

Levansucrases (LS) are fructosyltransferases (FTFs) belonging to family 68 of glycoside hydrolases (GH68) using sucrose as substrate to synthesize levan, a fructose polymer. From a multiple sequence analysis of GH68 family proteins, nine residues were selected and their role in acceptor and product specificity, as well as in biochemical Bacillus subtilis LS properties, was investigated. A product specificity modification was obtained with mutants Y429N and R433A that no longer produce levan but exclusively oligosaccharides. An effect of the mutation S164A was observed on enzyme stability and kinetic behavior; this mutation also induces a levan activation effect that enhances the reaction rate. We report the crystallographic structure of this mutant and found that S164 is an important residue to maintain the nucleophile position in the active site. We also found evidence of the important role of Y429 in acceptor specificity: this is a key residue coordinating the sucrose position in the catalytic domain-binding pocket. Some of these mutations resulted in LS with a broad range of specificities and new biochemical properties.

Enzymatic hydrolysis of fructans in the tequila production process.

In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequilas organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.

Size product modulation by enzyme concentration reveals two distinct levan elongation mechanisms in Bacillus subtilis levansucrase.

Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a high-molecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a non-processive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ∼70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift.

Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB).

Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.

Fructooligosaccharide production by a truncated Leuconostoc citreum inulosucrase mutant

Abstract Site-directed mutagenesis was performed on IslA4, a truncated form of inulosucrase (IS) derived from Leuconostoc citreum IS that contains only the IS catalytic domain. This truncated form is more hydrolytic than the wild-type enzyme and produces both high-molecular-weight inulin and fructooligosaccharides (FOS). Among the various mutants obtained from IslA4 by following strategies designed for SacB (the levansucrase from Bacillus subtilis), S425A no longer produces inulin, but instead produces FOS exclusively and hydrolyzes sucrose. Reaction conditions were explored to increase FOS productivity by reducing the hydrolysis, resulting in 65% conversion from a 0.67 M sucrose solution. S425A displays complex kinetic behavior in which the transfructosylation rate is described by first-order kinetics, while sucrose hydrolysis follows Michaelis–Menten behavior. This combined model correctly describes both the overall initial reaction rate as well as the reaction evolution for FOS synthesis. S425A IS may be useful for the synthesis of FOS from sucrose.