Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Asymmetric Synthesis of N-Substituted α-Amino Esters from α-Ketoesters via Imine Reductase-Catalyzed Reductive Amination.

N-Substituted α-amino esters are widely used as chiral intermediates in a range of pharmaceuticals. Here we report the enantioselective biocatalyic synthesis of N-substituted α-amino esters through the direct reductive coupling of α-ketoesters and amines employing sequence diverse metagenomic imine reductases (IREDs). Both enantiomers of N-substituted α-amino esters were obtained with high conversion and excellent enantioselectivity under mild reaction conditions. In addition >20 different preparative scale transformations were performed highlighting the scalability of this system.

Photo-biocatalytic One-Pot Cascades for the Enantioselective Synthesis of 1,3-Mercaptoalkanol Volatile Sulfur Compounds.

The synthesis of enantiomerically pure 1,3-mercaptoalkanol volatile sulfur compounds through a one-pot photo-biocatalytic cascade reaction is described. Two new KRED biocatalysts with opposite enantioselectivity were discovered and proved to be efficient on a wide range of substrates. The one-pot cascade reaction combining photocatalytic thio-Michael addition with biocatalytic ketoreduction in an aqueous medium provides a green and sustainable approach to enantiomerically pure 1,3-mercaptoalkanols in high yields with excellent enantioselectivity.

Rhodococcus jostii: a home for Rhodococcus strain RHA1.

The taxonomic position of Rhodococcus strain RHA1, an effective degrader of polychlorinated biphenyls with a large linear chromosome, was established using a polyphasic approach. The morphological and chemotaxonomic properties of the strain were typical of members of the genus Rhodococcus. The strain shared a high level of 16S rRNA sequence similarity (99.9 %) with the type strain of Rhodococcus jostii, a member of the Rhodococcus erythropolis subclade. The two strains shared a DNA:DNA relatedness value well above the cut-off point recommended for the circumscription of genomic species and had a broad range of phenotypic properties in common. The combination of genomic and phenotypic data show strain RHA1 to be a bona fide member of the species Rhodococcus jostii.

Analysis of the microbial community and geochemistry of a sediment core from Great Slave Lake, Canada.

Sediment cores taken from Great Slave Lake, Canada, were analysed to investigate their metabolically active microbial populations and geochemistry. The amplification of cDNA detected metabolically active bacterial (50 separate bands) and archaeal (49 separate band) communities. The bacterial communities were further resolved indicating active actinobacterial and γ-proteobacterial communities (36 and 43 individual bands respectively). Redundancy discriminate analysis and Monte Carlo permutation testing demonstrated the significant impact of geochemical parameters on microbial community structures. Geochemical analyses suggest that the upper 0.4 m represents soil weathering and erosion in the lake catchment. An increase in organic carbon in the lower core suggests either more primary productivity, indicating warmer climate conditions, associated with Holocene Climatic Optimum conditions pre 5,000 years BP or change from a reducing environment in the lower core to an oxidizing environment during more recent deposition. Drivers for bacterial, archaeal and actinobacterial community structures were sediment particle size, and its mineral composition. Depth also significantly affected γ- proteobacterial community structure. In contrast the organic carbon content did not significantly shape the microbial community structures within the sediment. This study indicates that geochemical parameters significantly contribute to microbial community structure in these sediments.