Numata, K. | bioRxiv Science

bioRxiv2017

Atomic resolution structure of serine protease proteinase K at ambient temperature

Atomic resolution structures (beyond 1.20 [A]) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 [A] resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

biochemistry↗

bioRxiv2016

Metabolomic and proteomic analyses of a quiescent Escherichia coli cell factory reveal the mechanisms behind its production efficiency

1Quiescent (Q-Cell) Escherichia coli cultures can be created by using the signalling molecule indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks. However, the reason for the increased productivity of cells in this state was previously unknown. We show here that Q-cells can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater per-cell productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Metabolomic data show that by disrupting the proton-motive force, indole interrupts the tricarboxylic acid cycle, leading to the accumulation of metabolites in the glycolysis pathway that are excellent starting points for high-value chemical production. By comparing protein expression patterns between wild-type and Q-cell cultures we show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g.L-1 of 3HB compared to 18.6 g.L-1 in the control. Therefore, Q-cells have high potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.

microbiology↗