Ultrahigh-Throughput Screening of Metagenomic Libraries Using Droplet Microfluidics.

Droplet microfluidics enables the ultrahigh-throughput screening of the natural or man-made genetic diversity for industrial enzymes, with reduced reagent consumption and lower costs than conventional robotic alternatives. Here we describe an example of metagenomic screening for nucleoside 2'-deoxyribosyl transferases using FACS as a more widespread and accessible alternative than microfluidic on-chip sorters. This protocol can be easily adapted to directed evolution libraries by replacing the library construction steps and to other enzyme activities, e.g., oxidases, by replacing the proposed coupled assay.

Highly Promiscuous Oxidases Discovered in the Bovine Rumen Microbiome.

The bovine rumen hosts a diverse microbiota, which is highly specialized in the degradation of lignocellulose. Ruminal bacteria, in particular, are well equipped to deconstruct plant cell wall polysaccharides. Nevertheless, their potential role in the breakdown of the lignin network has never been investigated. In this study, we used functional metagenomics to identify bacterial redox enzymes acting on polyaromatic compounds. A new methodology was developed to explore the potential of uncultured microbes to degrade lignin derivatives, namely kraft lignin and lignosulfonate. From a fosmid library covering 0.7 Gb of metagenomic DNA, three hit clones were identified, producing enzymes able to oxidize a wide variety of polyaromatic compounds without the need for the addition of copper, manganese, or mediators. These promiscuous redox enzymes could thus be of potential interest both in plant biomass refining and dye remediation. The enzymes were derived from uncultured Clostridia, and belong to complex gene clusters involving proteins of different functional types, including hemicellulases, which likely work in synergy to produce substrate degradation.

Directed evolution of the bacterial endo-ß-1,4-glucanase from Streptomyces sp. G12 towards improved catalysts for lignocellulose conversion.

With the aim to develop biocatalysts for enhanced hydrolysis of (hemi)cellulose into monosaccharides, random diversity by directed evolution was introduced in the gene coding for the endo-ß-1,4-glucanase from Streptomyces sp. G12 which had been recombinantly expressed in Escherichia coli and named rCelStrep. The main objectives were therefore to set up a complete strategy for creation and automated screening of rCelStrep evolved direct mutants and to apply it to generate and screen a library of 10,000 random mutants to select the most active variants. The diversity was introduced in the gene by error-prone polymerase chain reaction. A primary qualitative screening on solid plates containing carboxymethylcellulose as the substrate allowed selecting 2200 active clones that were then subjected to a secondary quantitative screening towards AZO-CMC for the selection of 76 improved variants that were cultured in flasks and characterized. Five rCelStrep mutants exhibiting the highest hydrolytic activities than the wild-type enzyme were further characterized and applied to the bioconversion of the pretreated Arundo donax lignocellulosic biomass. It is worth of noting that one of the five tested mutants exhibited a 30% improvement in bioconversion yields compared to the wild-type enzyme, despite the absence of the carbohydrate binding module domain in this variant. Homology models of the three-dimensional structures of the catalytic and binding modules of rCelStrep were obtained and localization of mutations on these models allowed us to speculate on the structure-function relationships of the mutants.

Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 ß-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria.

The ß-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues Asp(393), Glu(416) and Glu(364) was performed. NMR analysis of the xyloside hydrolysis products, the change of the reaction rate-limiting step for the Glu(416) mutants, the pH dependency of E416A activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining mechanism of glycoside hydrolysis, Glu(416) playing the role of acid/base catalyst and Asp(393) that of nucleophile.

Role of glycoside phosphorylases in mannose foraging by human gut bacteria.

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze ß-D-Manp-1,4-ß-D-GlcpNAc-1,4-D-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.

Functional metagenomics reveals novel pathways of prebiotic breakdown by human gut bacteria.

The human intestine hosts a complex bacterial community that plays a major role in nutrition and in maintaining human health. A functional metagenomic approach was used to explore the prebiotic breakdown potential of human gut bacteria, including non-cultivated ones. Two metagenomic libraries, constructed from ileum mucosa and fecal microbiota, were screened for hydrolytic activities on the prebiotic carbohydrates inulin, fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and lactulose. The DNA inserts of 17 clones, selected from the 167 hits that were identified, were pyrosequenced in-depth, yielding in total 407, 420 bp of metagenomic DNA. From these sequences, we discovered novel prebiotic degradation pathways containing carbohydrate transporters and hydrolysing enzymes, for which we provided the first experimental proof of function. Twenty of these proteins are encoded by genes that are also present in the gut metagenome of at least 100 subjects, whatever are their ages or their geographical origin. The sequence taxonomic assignment indicated that still unknown bacteria, for which neither culture conditions nor genome sequence are available, possess the enzymatic machinery to hydrolyse the prebiotic carbohydrates tested. The results expand the vision on how prebiotics are metabolized along the intestine, and open new perspectives for the design of functional foods.

Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst.

The mutant penicillin G acylase (PGA) 3K-PGA contains three additional Lys residues on the surface opposite the active site. This protein was designed to selectively drive its immobilization on aldehyde supports. We describe here a modified bottom-up proteomic method to assess the orientation of the immobilized wild-type and mutant proteins to verify our hypothesis of a driven immobilization induced by the mutations introduced. Tryptic digestion of the immobilized enzymes followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of released peptides was performed. This protocol generated peptides from the most accessible surface areas of the immobilized protein, thus not directly bound to the solid support, providing direct evidence of the areas involved in the linkage to the solid matrix. The results obtained suggest that 72 % of the wild-type PGA is immobilized on aldehyde agarose mainly through the Lys residues on the same side of the active site, whereas 3K-PGA reacted with the same support preferentially through the additional Lys residues introduced by mutation on the opposite side. This demonstrates that the active site of the 3K-PGA faces mostly (63 %) toward the reaction medium, resulting in significantly improved accessibility to the substrates. This finding is supported by the catalytic properties of the immobilized biocatalysts. The two immobilized preparations were tested in the synthesis of mandelyl-7-aminocephalosporanic acid (mandelyl-7-ACA) by N-acylation of the ß-lactam nucleus (7-aminocephalosporanic acid) with mandelic acid methyl ester: upon immobilization, the synthetic properties of wild-type PGA strongly decreased, whereas those of 3K-PGA were unaffected. Furthermore, the activity of 3K-PGA was not influenced by the physicochemical nature of the support used for immobilization (glyoxyl agarose or aldehyde Sepabeads) unlike that of wild-type PGA, whose active site is close to the matrix. The results obtained from the analytical characterization correlate well with those obtained by investigation of the synthetic properties of the immobilized enzymes both in the synthesis of mandelyl-7-ACA and in the preparative synthesis of cefazolin. This work highlights the effect exerted by site-directed mutagenesis on the orientation of PGA upon immobilization on solid matrices and suggests how protein engineering tools can be exploited in a synergistic fashion to rationally develop efficient biocatalysts.

Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase.

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH2 group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and ß24 (ßF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-ßF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-ßF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-ßF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized ßF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.

New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase.

BACKGROUND: Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic beta-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem. RESULTS: Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell. CONCLUSION: The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues on a predetermined region of the enzyme. The newly designed biocatalysts display improved synthetic performances and are able to maintain a similar activity to the free enzymes. Finally, we found that the activity of the immobilized enzyme proportionally improves with the number of introduced Lys.