Tailor-made poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), which is produced by several Bacillus species, is a chiral biopolymer composed of D- and L-glutamate monomers and has various industrial applications. However, synthesized γ-PGA exhibits great structural diversity, and the structure must be controlled to broaden its industrial use. The biochemical pathways for γ-PGA production suggest that the polymer properties molecular weight (MW) and stereochemical composition are influenced by (1) the affinity of γ-PGA synthetase for the two alternative glutamate enantiomers and (2) glutamate racemase activity; hence, the availability of the monomers. In this study, we report tailor-made γ-PGA synthesis with B. subtilis by combining PGA synthetase and glutamate racemase genes from several Bacillus strains. The production of structurally diverse γ-PGA was thereby achieved. Depending on the PGA synthetase and glutamate racemase origins, the synthesized γ-PGA contained 3-60% D-glutamate. The exchange of PGA synthetase changed the MW from 40 to 8500 kDa. The results demonstrate the production of low-, medium-, and high-MW γ-PGA with the same microbial chassis.

Microbial single-cell analysis in picoliter-sized batch cultivation chambers.

Microfluidics has enabled various research projects in the field of microbial single-cell analysis. In particular, single-use microfluidic cultivation devices combined with automated time-lapse imaging provide powerful approaches for analyzing microbial phenomena at the single-cell level. High spatiotemporal resolution facilitates individual cell identification and tracking, delivering detailed insights into areas like phenotypic population heterogeneity, which can be highly relevant to the fate of a microbial population and may negatively impact the efficiency of biotechnological fermentations. New tools need to be developed to access the origin of population heterogeneity and understand its functional role. In this study, we present a microfluidic device for batch cultivations inside picoliter-sized cultivation chambers that can be reversibly isolated from continuous medium supply. Therefore, the cultivation broth is simply replaced by a continuous flow of humidified air, removing any medium residue along the supply channels but preserving five picoliters of cultivation medium inside the cultivation chambers in a highly parallel manner. Living cells can grow inside our microfabricated batch chambers, which can accommodate up to several hundred cells. The chamber height approximately matches the diameter of a single cell, facilitating cell growth in monolayers that are ideal for image-based cell analysis. We successfully demonstrated the growth of Escherichia coli during continuous medium perfusion and batch cultivation conditions. As expected, the cells grew exponentially under continuous medium influx until the maximum chamber capacity was reached, but when they were cultivated under batch conditions, cellular growth underwent an exponential phase, followed by a stationary phase with obvious morphological changes.