Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is a Gram-positive, non-pathogenic soil bacterium that has been extensively investigated for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers were restricted due to limited genetic accessibility of the organism up to now. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants were generated and compared in batch fermentations for the first time. Downstream processing was facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Spore formation was inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of P. polymyxa. Optimization of the carbon flux towards 2,3-butanediol was achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain showed 45 % increased productivity, reaching a final concentration of 43.8 g L-1 butanediol. A yield of 0.43 g g-1 glucose was achieved, accounting for 86 % of the theoretical maximum.

Design and characterization of a modular membrane protein anchor to functionalize the moss Physcomitrella patens with extracellular catalytic and/or binding activities.

Heterologous enzymes and binding proteins were secreted by the moss Physcomitrella patens or anchored extracellularly on its cell membrane in order to functionalize the apoplast as a biochemical reaction compartment. This modular membrane anchoring system utilizes the signal peptide and the transmembrane segment of the somatic embryogenesis receptor-like kinase (SERK), which were identified in a comprehensive bioinformatic analysis of the P. patens genome. By fusing the soluble enzyme NanoLuc luciferase to the signal peptide, its secretion capability was confirmed in vivo. The membrane localization of hybrid proteins comprising the SERK signal peptide, NanoLuc or other functional modules, the SERK transmembrane anchor, and a C-terminal GFP reporter was demonstrated using fluorescence microscopy as well as site-specific proteolytic release of the extracellular enzyme domain. Our membrane anchoring system enables the expression of various functional proteins in the apoplast of P. patens, empowering this photoautotrophic organism for biotechnological applications.