Development Of Whole-cell Biocatalysts Research Articles

Efficient recovery of beta-mannanase fused with LysM3014 domain in Escherichia coliby glass bead based-cell disruption method

Toward the development of whole cell biocatalysts via non-GMO strategy, in this study, three cell disruption methods (sonication, freezingand thawing and bead-shaking) have been evaluatedto recover efficiently fusion protein LysM3014-ManB containing a beta-mannanase (ManB) from Bacillus lichenifomisfused with a single LysM3014 domain derived from a putative extracellulartransglycosylase Lp_3014 of Lactobacillus plantarumWCFS1. The results showed that the highest volumetric activity of fusion protein (~46651 U/lfermentation) was obtained when shaking the recombinant E. coliwith glass bead. Moreover, the enzymatic activity of LysM3014-ManB increased approximately 1.4 folds(~66000 U/lfermentation)under the optimum conditions of glass bead-based cell disruption (falcon 50 ml, glass bead ratio of 1.0 g/mland disruption time of 8 min). The study indicates that shaking the cells with glass bead is likely to be the simplest and the most efficient cell disruption methodin comparison with other disruption methods.

Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

Background Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum.ResultsManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. ConclusionThis study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially ‘prebiotic’ oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.

Cell surface and cellular debris-associated heat-stable lipolytic enzyme activities of the marine alga Nannochloropsis oceanica

Microbial surface display of lipases can be effectively employed for the development of whole-cell biocatalysts for industrial bioconversions. In the present work, we report for the first time the presence of thermostable lipolytic enzyme activities against p-nitrophenyl laurate, both on the cell surface and the cellular debris fraction of the marine microalga Nannochloropsis oceanica (strain CCMP1779). Whole cell-associated lipolytic activity (WCLA) shows a 2.5-fold stimulation after heat treatment at 100 °C for 60 min, while the activity of the respective cell debris is retained for 15 min. In contrast, heat treatment renders the soluble fraction of the disrupted cells inactive. The progress curve of cellular debris-associated lipase activity is biphasic and levels off very fast. Treatment with the surfactants SDS, Triton X-100 and CHAPS, which are known to inhibit lipase activity in various degrees, results in a loss of both cell bound and cell debris lipolytic activities (CDLA). The highest whole cell lipase catalytic efficiency was observed against p-nitrophenyl butyrate and the optimum pH for hydrolysis was determined at pH 7.0. Both unheated and heated undisrupted whole cell biocatalysts are also catalytically active against olive oil. High-salt concentrations (1M NaCl) lead to about 50% whole cell enzyme inhibition whereas the activity of heated cells increases. These findings offer novel insight into the biocatalytic properties and the biotechnological applicability of microalgal lipases from N. oceanica.

Impacts and perspectives of prenyltransferases of the DMATS superfamily for use in biotechnology.

Prenylated compounds are ubiquitously found in nature and demonstrate interesting biological and pharmacological activities. Prenyltransferases catalyze the attachment of prenyl moieties from different prenyl donors to various acceptors and contribute significantly to the structural and biological diversity of natural products. In the last decade, significant progress has been achieved for the prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily. More than 40 members of these soluble enzymes are identified in microorganisms and characterized biochemically. These enzymes were also successfully used for production of a large number of prenylated derivatives. N1-, C4-, C5-, C6-, and C7-prenylated tryptophan and N1-, C2-, C3-, C4-, and C7-prenylated tryptophan-containing peptides were obtained by using DMATS enzymes as biocatalysts. Tyrosine and xanthone prenyltransferases were used for production of prenylated derivatives of their analogs. More interestingly, the members of the DMATS superfamily demonstrated intriguing substrate and catalytic promiscuity and also used structurally quite different compounds as prenyl acceptors. Prenylated hydroxynaphthalenes, flavonoids, indolocarbazoles, and acylphloroglucinols, which are typical bacterial or plant metabolites, were produced by using several fungal DMATS enzymes. Furthermore, the potential usage of these enzymes was further expanded by using natural or unnatural DMAPP analogs as well as by coexpression with other genes like NRPS and by development of whole cell biocatalyst.

Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam.

At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30°C other than 90°C, with a substantial decrease of 60°C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100g racemic vince lactam to produce 49.2g (-) vince lactam at 30°C within 4h. By using chiral HPLC, the ee value of the produced (-) vince lactam was determined to be 99.5%. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (-) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.

Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.

Optimisation of surface expression using the AIDA autotransporter

BackgroundBacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.ResultsThe use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface-anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l-1 could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.ConclusionsA number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

Novel auto-inducing expression systems for the development of whole-cell biocatalysts

Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of (S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes.

Enantioselective biocatalysis optimized by directed evolution.

Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.