Fine tuning Exo2, a small molecule inhibitor of secretion and retrograde trafficking pathways in mammalian cells

The small molecule 4-hydroxy-3-methoxybenzaldehyde (5,6,7,8-tetrahydro[1]benzothieno[2,3- d]pyrimidin-4-yl)hydrazone (Exo2) stimulates morphological changes at the mammalian Golgi and trans-Golgi network that are virtually indistinguishable from those induced by brefeldin A. Both brefeldin A and Exo2 protect cells from intoxication by Shiga(-like) toxins by acting on other targets that operate at the early endosome, but do so at the cost of high toxicity to target cells. The advantage of Exo2 is that it is much more amenable to chemical modification and here we report a range of Exo2 analogues produced by modifying the tetrahydrobenzothienopyrimidine core, the vanillin moiety and the hydrazone bond that links these two. These compounds were examined for the morphological changes they stimulated at the Golgi stack, the trans Golgi network and the transferrin receptor-positive early endosomes and this activity correlated with their inherent toxicity towards the protein manufacturing ability of the cell and their protective effect against toxin challenge. We have developed derivatives that can separate organelle morphology, target specificity, innate toxicity and toxin protection. Our results provide unique compounds with low toxicity and enhanced specificity to unpick the complexity of membrane trafficking networks

C1 compounds as auxiliary substrate for engineered Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C1 compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host