High level production of flavonoid rhamnosides by metagenome-derived Glycosyltransferase C in Escherichia coli utilizing dextrins of starch as a single carbon source.

Flavonoids exert a wide variety of biological functions that are highly attractive for the pharmaceutical and healthcare industries. However, their application is often limited by low water solubility and poor bioavailability, which can generally be relieved through glycosylation. Glycosyltransferase C (GtfC), a metagenome-derived, bacterial glycosyltransferase, was used to produce novel and rare rhamnosides of various flavonoids, including chrysin, diosmetin, biochanin A, and hesperetin. Some of them are to our knowledge firstly described within this work. In our study we deployed a new metabolic engineering approach to increase the rhamnosylation rate in Escherichia coli whole cell biotransformations. The coupling of maltodextrin metabolism to glycosylation was developed in E. coli MG1655 with the model substrate hesperetin. The process proved to be highly dependent on the availability of maltodextrins. Maximal production was achieved by the deletion of the phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridyltransferase (galU) genes and simultaneous overexpression of the dTDP-rhamnose synthesis genes (rmlABCD) as well as glucan 1,4-alpha-maltohexaosidase for increased maltodextrin degradation next to GtfC in E. coli UHH_CR5-A. These modifications resulted in a 3.2-fold increase of hesperetin rhamnosides compared to E. coli MG1655 expressing GtfC in 24 h batch fermentations. Furthermore, E. coli UHH-CR_5-A was able to produce a final product titer of 2.4 g/L of hesperetin-3'-O-rhamnoside after 48 h. To show the versatility of the engineered E. coli strain, biotransformations of quercetin and kaempferol were performed, leading to production of 4.3 g/L quercitrin and 1.9 g/L afzelin in a 48 h time period, respectively. So far, these are the highest published yields of flavonoid rhamnosylation using a biotransformation approach. These results clearly demonstrate the high potential of the engineered E. coli production host as a platform for the high level biotransformation of flavonoid rhamnosides.

Metagenomic cellulases highly tolerant towards the presence of ionic liquids--linking thermostability and halotolerance.

Cellulose is an important renewable resource for the production of bioethanol and other valuable compounds. Several ionic liquids (ILs) have been described to dissolve water-insoluble cellulose and/or wood. Therefore, ILs would provide a suitable reaction medium for the enzymatic hydrolysis of cellulose if cellulases were active and stable in the presence of high IL concentrations. For the discovery of novel bacterial enzymes with elevated stability in ILs, metagenomic libraries from three different hydrolytic communities (i.e. an enrichment culture inoculated with an extract of the shipworm Teredo navalis, a biogas plant sample and elephant faeces) were constructed and screened. Altogether, 14 cellulolytic clones were identified and subsequently assayed in the presence of six different ILs. The most promising enzymes, CelA2, CelA3 (both derived from the biogas plant) and CelA84 (derived from elephant faeces), showed high activities (up to 6.4 U/mg) in the presence of 30% (v/v) ILs. As these enzymes were moderately thermophilic and halotolerant, they retained 40% to 80% relative activity after 34 days in 4 M NaCl, and they were benchmarked with two thermostable enzymes, CelA from Thermotoga maritima and Cel5K from a metagenome library derived from Avachinsky crater in Kamchatka. These enzymes also exhibited high activity (up to 11.1 U/mg) in aqueous IL solutions (30% (v/v)). Some of the enzymes furthermore exhibited remarkable stability in 60% (v/v) IL. After 4 days, CelA3 and Cel5K retained up to 79% and 100% of their activity, respectively. Altogether, the obtained data suggest that IL tolerance appears to correlate with thermophilicity and halotolerance.

Screening Glycosyltransferases for Polyphenol Modifications.

Glycosyltransferases offer the opportunity to glycosylate a variety of substrates including health beneficial molecules like flavonoids in a regiospecific manner. Flavonoids are plant secondary metabolites that have antimicrobial, antioxidative, and health beneficial effects. Glycosylation often has impact on these properties and furthermore enhances the water solubility, the stability, and the bioavailability of the molecules. To detect flavonoid glycosylating enzymes we established a metagenome screen for the discovery of modifying clones. This function based screening technique can furthermore detect other modifications like methylations. The method relies on analysis of the culture supernatant extracts from biotransformation reactions in a thin layer chromatography (TLC) approach.

Screening for Cellulase Encoding Clones in Metagenomic Libraries.

For modern biotechnology there is a steady need to identify novel enzymes. In biotechnological applications, however, enzymes often must function under extreme and nonnatural conditions (i.e., in the presence of solvents, high temperature and/or at extreme pH values). Cellulases have many industrial applications from the generation of bioethanol, a realistic long-term energy source, to the finishing of textiles. These industrial processes require cellulolytic activity under a wide range of pH, temperature, and ionic conditions, and they are usually carried out by mixtures of cellulases. Investigation of the broad diversity of cellulolytic enzymes involved in the natural degradation of cellulose is necessary for optimizing these processes.

Deep metagenome and metatranscriptome analyses of microbial communities affiliated with an industrial biogas fermenter, a cow rumen, and elephant feces reveal major differences in carbohydrate hydrolysis strategies.

BACKGROUND: The diverse microbial communities in agricultural biogas fermenters are assumed to be well adapted for the anaerobic transformation of plant biomass to methane. Compared to natural systems, biogas reactors are limited in their hydrolytic potential. The reasons for this are not understood. RESULTS: In this paper, we show that a typical industrial biogas reactor fed with maize silage, cow manure, and chicken manure has relatively lower hydrolysis rates compared to feces samples from herbivores. We provide evidence that on average, 2.5 genes encoding cellulolytic GHs/Mbp were identified in the biogas fermenter compared to 3.8 in the elephant feces and 3.2 in the cow rumen data sets. The ratio of genes coding for cellulolytic GH enzymes affiliated with the Firmicutes versus the Bacteroidetes was 2.8:1 in the biogas fermenter compared to 1:1 in the elephant feces and 1.4:1 in the cow rumen sample. Furthermore, RNA-Seq data indicated that highly transcribed cellulases in the biogas fermenter were four times more often affiliated with the Firmicutes compared to the Bacteroidetes, while an equal distribution of these enzymes was observed in the elephant feces sample. CONCLUSIONS: Our data indicate that a relatively lower abundance of bacteria affiliated with the phylum of Bacteroidetes and, to some extent, Fibrobacteres is associated with a decreased richness of predicted lignocellulolytic enzymes in biogas fermenters. This difference can be attributed to a partial lack of genes coding for cellulolytic GH enzymes derived from bacteria which are affiliated with the Fibrobacteres and, especially, the Bacteroidetes. The partial deficiency of these genes implies a potentially important limitation in the biogas fermenter with regard to the initial hydrolysis of biomass. Based on these findings, we speculate that increasing the members of Bacteroidetes and Fibrobacteres in biogas fermenters will most likely result in an increased hydrolytic performance.

A comparative metagenome survey of the fecal microbiota of a breast- and a plant-fed Asian elephant reveals an unexpectedly high diversity of glycoside hydrolase family enzymes.

A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

Screening for cellulases with industrial value and their use in biomass conversion.

Cellulose is an easily renewable and highly occurring resource. To take advantage of this great potential, there is a constant need of new cellulose degrading enzymes. In industrial applications enzymes have to function under extreme conditions like high temperature, very acidic or basic pH and different solvents. Cellulases have a huge area of application, for example the textile and food industry as well as the generation of bioethanol as an alternative energy source. They have the ability to yield a great energetic potential, but there is still a lack of economical technologies to conquer the stability of the cellulose structure. Via metagenomic research and well-directed screening, it is possible to detect new cellulases, which are active under tough industrial conditions.

Screening for cellulase-encoding clones in metagenomic libraries.

Modern biotechnology has the steady need to continuously identify novel enzymes for use in biotechnological applications. In industrial applications, however, enzymes often have to function under extreme and nonnatural conditions (i.e., in the presence of solvents, high temperature and/or at extreme pH values). Cellulases have many industrial applications from the generation of bioethanol, a realistic long-term energy source, to the finishing of textiles. These industrial processes require cellulolytic activity under a range of pH, temperature, and ionic conditions, and they are usually carried out by mixtures of cellulases. Investigation of the broad diversity of cellulolytic enzymes involved in the natural degradation of cellulose is necessary for optimization of these processes.