Jürgen Eck

Metagenomics and industrial applications

Different industries have different motivations to probe the enormous resource that is uncultivated microbial diversity. Currently, there is a global political drive to promote white (industrial) biotechnology as a central feature of the sustainable economic future of modern industrialized societies. This requires the development of novel enzymes, processes, products and applications. Metagenomics promises to provide new molecules with diverse functions, but ultimately, expression systems are required for any new enzymes and bioactive molecules to become an economic success. This review highlights industrial efforts and achievements in metagenomics.

Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics

Acidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNA‐based molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210 000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co‐linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine‐DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two‐component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well‐studied bacterial phyla.

Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases.

In the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non‐redundant subtilase sequence tags with degenerate primers. Furthermore, an activity‐ as well as a sequence homology‐based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full‐length protease genes were recovered, sharing only 37–85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin‐like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications.

Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome-derived esterases.

Enantiomerically pure β‐arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate‐based screening using a pH‐indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.

The Impact of Non-cultivated Biodiversity on Enzyme Discovery and Evolution

Abstract The search for novel enzymes with biotechnological potential in the fine chemical, food and feed, detergent and cosmetics industries is driven by the need to improve existing processes and applications, to design novel processes for innovative products or intermediates or to avoid intellectual property related operative restrictions. Strategies for obtaining novel biocatalysts will be based on screening natural biodiversity or a combination of nature derived scaffolds and optimization by directed evolution technology. Considering the enormous potential of in vitro mutational and recombinatorial strategies to alter genes and improve enzyme properties, we propose that it might be advantageous to select improved molecular starting points before embarking on the arduous walk through sequence space towards optimized performance

The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis

Bacillus subtilis is a commonly used host for the heterologous expression of genes in academia and industry. Many factors are known to influence the expression yield in this organism e.g. the complementarity between the Shine-Dalgarno sequence (SD) and the 16S-rRNA or secondary structures in the translation initiation region of the transcript. In this study, we analysed the impact of the nucleotide composition between the SD sequence and the start codon (the spacer sequence) on the expression yield. We demonstrated that a polyadenylate-moiety spacer sequence moderately increases the expression level of laccase CotA from B. subtilis. By screening a library of artificially generated spacer variants, we identified clones with greatly increased expression levels of two model enzymes, the laccase CotA from B. subtilis (11 fold) and the metagenome derived protease H149 (30 fold). Furthermore, we demonstrated that the effect of the spacer sequence is specific to the gene of interest. These results prove the high impact of the spacer sequence on the expression yield in B. subtilis.

Now we are talking sense! Functional approaches to novel nutraceuticals and cosmeceuticals

For competitive and certainly for economical reasons the pharmaceutical, food and cosmetic industries strive for the development and manufacturing of beneficial bioactive consumer products. Whereas bioactivity is well accepted for pharmaceuticals, it often provokes a conflict for the food and cosmetic industries, because their original purpose strictly speaking was either providing pure caloric respectively decorative effects; an aspect that is supported by the official regulatory view up to now. The consumer who buys products for personal and non-professional use demands more and more effective bioactive products to improve his personal wellness and state of health. However, defining the term bioactive as “interacting with and/or acting on human living tissue and a molecular target therein”, a good part of foods and cosmetics are and have been bioactive all the time. In terms of bioactive product development, the situation has dramatically changed in the last decade as human molecular targets have become accessible through the human genome sequencing projects. Thus, targets which before have mostly been identified by time-consuming and expensive reverse genetics in pharmaceutical research now become available by rational, sequenceand target gene-dependent screening approaches.This opens up interesting perspectives for the food and cosmetic industries as the screening technologies originally developed for pharmaceutical research are now not only available but also affordable for these non-pharmaceutical industries, which traditionally have a much lower profit volume. Through the increasing interest in preventive healthcare and wellness within the industrial world over the recent years, the pharmaceutical, food and cosmetic industries have created new terms for the marketing of healthcare products with claimed medicinalor drug-like benefits. In the context of recent developments towards novel human molecular targets, we review and redefine emerging terms for bioactive cosmetic and food ingredients, namely nutraceuticals, nutracosmeceuticals and cosmeceuticals (Box 1). Foods claiming to have a medicinal effect on human health are called “functional food” or in the Asian world “food of specified health use” (Foshu). There is no universally accepted definition for functional food, but it is most often defined as food containing a bioactive nutraceutical – a semantical blend of nutrition and pharmaceutical – that provides a health benefit beyond the basic nutritional function and in particular protects against chronic diseases. This definition separates nutraceuticals from dietary supplements like vitamins, minerals, herbs/botanicals, amino acids and medical foods mainly intended for specific dietary management. Prominent examples for nutraceuticals are plantderived sterol esters or milk-derived peptides with the biological function to lower cholesterol levels. Biotech Highlight

Cytotoxic activity of recombinant bFGF-rViscumin fusion proteins

A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized. bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM. rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM. Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure. The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM. Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB. We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development.