Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

Secreted production of self-assembling peptides in Pichia pastoris by fusion to an artificial highly hydrophilic protein.

The undecapeptides CH(3)CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH(2) (P(11)-2) and CH(3)CO-Gln-Gln-Orn-Phe-Orn-Trp-Orn-Phe-Orn-Gln-Gln-NH(2) (P(11)-14) have unique self-assembly characteristics and broad application potential. Originally, these peptides were produced by chemical synthesis, which is costly and difficult to scale up to industrial levels in an economically feasible way. This article describes the efficient secreted production of these peptides (with free termini and ornithines replaced with lysines) in the methylotrophic yeast Pichia pastoris. The peptides were produced as enterokinase-cleavable fusions to the C-terminus of an artificial Solubility-Enhancing Protein (SEP). In vitro, the fused highly hydrophilic SEP proved to prevent self-assembly of the peptides. The SEP domain also facilitates product detection and allows convenient separation of the fusion protein from the broth by simple salt precipitation. After cleavage of the purified fusion protein with enterokinase, the free undecapeptides were obtained and P(11)-2 spontaneously assembled into a self-supporting gel, as intended. The properties of the SEP carrier could be advantageous for the production of other peptides.

Precision gels from collagen-inspired triblock copolymers.

Gelatin hydrogels find broad medical application. The current materials, however, are from animal sources, and their molecular structure and thermal properties cannot be controlled. This study describes recombinant gelatin-like polymers with a general design that inherently offers independent tuning of the cross-link density, melting temperature, and biocompatibility of the gel. The polymers contain small blocks with thermoreversible trimerization capacity and defined melting temperature, separated by hydrophilic nontrimerizing blocks defining the distance between the knot-forming domains. As an example, we report the secreted production in yeast at several g/L of two nonhydroxylated approximately 42 kDa triblock copolymers with terminal trimerizing blocks. Because only the end blocks formed cross-links, the molecular architecture of the gels is much more defined than that of traditional gelatins. The novel hydrogels had a approximately 37 degrees C melting temperature, and the dynamic elasticity was independent of the thermal history. The concept allows to produce custom-made precision gels for biomedical applications.