Mechanistic Modeling of In Vivo Translation in Reliably Identifies Well-Adapted and Optimized RNA Sequences.

Translation elongation is a multifaceted process that intricately links translational resource availability to the biophysical effects arising from the interaction of mRNA sequences, ribosomes, and nascent polypeptide chains. Optimizing (heterologous) gene expression via codon usage or tRNA preference alone may yield suboptimal outcomes. In this study, we present a comprehensive mechanistic model that accounts for the competition of tRNAs at the ribosomal A-site, internal Shine-Dalgarno sequence interactions, and the decelerating effects of positively charged peptide patches.

Protein secretion zones during overexpression of amylase within the Gram-positive cell wall.

Background: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B.

Growth-Rate Dependent Regulation of tRNA Level and Charging in Bacillus licheniformis.

Cellular growth crucially depends on protein synthesis and the abundance of translational components. Among them, aminoacyl-tRNAs play a central role in biosynthesis and shape the kinetics of mRNA translation, thus influencing protein production. Here, we used microarray-based approaches to determine the charging levels and tRNA abundance of Bacillus licheniformis.

Optimization of translation profiles enhances protein expression and solubility.

mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli.

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.

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.

Updating the metagenomics toolbox.

Metagenomics--the application of the genomics suit of technologies to uncultivated microorganisms--is coming of age. Sophisticated technologies are being developed and adapted to this promising genetic resource to make increasing use of the seemingly boundless molecular and functional diversity. Particular progress has been made in the areas of randomly proliferating limited-source DNA, massively parallel sequencing without cloning, isolating specific target sequences from highly complex template mixtures, high-throughput assay systems targeting metabolic pathways, artificial transcriptional regulators activating reporter genes to indicate enzymatic substrate conversion and cDNA cloning from extracted mRNA to directly clone actively expressed genes from a microbial consortium.

Metagenomics: an inexhaustible access to nature's diversity.

The chemical industry has an enormous need for innovation. To save resources, energy and time, currently more and more established chemical processes are being switched to biotechnological routes. This requires white biotechnology to discover and develop novel enzymes, biocatalysts and applications.

Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space.

Historically, biotechnology has missed up to 99% of existing microbial resources by using traditional screening techniques. Strategies of directly cloning 'environmental DNA' comprising the genetic blueprints of entire microbial consortia (the so-called 'metagenome') provide molecular sequence space that along with ingenious in vitro evolution technologies will act synergistically to bring a maximum of available sequence-space into biocatalytic application.