Enlarging the synthetic biology toolbox for Pichia pastoris: Golden Gate cloning and CRISPR/Cas9

State-of-the-art strain engineering techniques for the protein producing yeast host Pichia pastoris include overexpression of homologous and heterologous genes, and deletion of host genes. For this purpose overexpression vectors and gene deletion methods such as the split marker technique have been established. For metabolic and cell engineering purposes, the simultaneous overexpression of more than one gene is often needed. Previous approaches employing subsequent steps of overexpression and marker recycling were time- and labor-consuming. Therefore, efficient systems allowing multiple gene overexpression are required, that can be stably integrated into the P. pastoris genome. To this end, we developed a synthetic biology toolbox based on Golden Gate cloning to enable efficient construction of complex and versatile over-expression vectors. Up to five different expression cassettes, employing a library of promoters and terminators can be combined into one vector, and successfully integrated into the genomic DNA of P. pastoris at targeted loci in one step. Recent trends in synthetic biology, however, go into the direction of building up large and complex reaction networks. To allow for clean and unscarred genetic engineering, a CRISPR/Cas9 based method for gene insertions, deletions and replacements was developed, which paves the way for precise genomic rearrangements in P. pastoris. By using this technique precise genomic integrations were performed efficiently without integrative selection markers. The repertoire of genetic techniques developed so far, will provide a wide variety of possibilities to engineer P. pastoris. Applications for these synthetic biology tools in cell engineering of recombinant P. pastoris will be presented

Nanomaterials by severe plastic deformation: review of historical developments and recent advances

International audienceSevere plastic deformation (SPD) is effective in producing bulk ultrafine-grained and nanostructured materials with large densities of lattice defects. This field, also known as NanoSPD, experienced a significant progress within the past two decades. Beside classic SPD methods such as high-pressure torsion, equal-channel angular pressing, accumulative roll-bonding, twist extrusion, and multi-directional forging, various continuous techniques were introduced to produce upscaled samples. Moreover, numerous alloys, glasses, semiconductors, ceramics, polymers, and their composites were processed. The SPD methods were used to synthesize new materials or to stabilize metastable phases with advanced mechanical and functional properties. High strength combined with high ductility, low/room-temperature superplasticity, creep resistance, hydrogen storage, photocatalytic hydrogen production, photocatalytic CO2 conversion, superconductivity, thermoelectric performance, radiation resistance, corrosion resistance, and biocompatibility are some highlighted properties of SPD-processed materials. This article reviews recent advances in the NanoSPD field and provides a brief history regarding its progress from the ancient times to modernity