Core and auxiliary functions of one-carbon metabolism in Pseudomonas putida exposed by a systems-level analysis of transcriptional and physiological responses

The soil bacterium Pseudomonas putida is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. P. putida is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde, and formate) oxidation-yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in P. putida. RNA sequencing identified two oxidoreductases, encoded by PP_0256 and PP_4596, transcriptionally active in the presence of formate. Quantitative physiology of deletion mutants revealed growth defects at high formate concentrations, pointing to an important role of these oxidoreductases in C1 tolerance. Moreover, we describe a concerted detoxification process for methanol and formaldehyde, the C1 intermediates upstream formate. Alcohol oxidation to highly-reactive formaldehyde by PedEH and other broad-substrate-range dehydrogenases underpinned the (apparent) suboptimal methanol tolerance of P. putida. Formaldehyde was mostly processed by a glutathione-dependent mechanism encoded in the frmAC operon, and thiol-independent FdhAB and AldB-II overtook detoxification at high aldehyde concentrations. Deletion strains were constructed and characterized towards unveiling these biochemical mechanisms, underscoring the worth of P. putida for emergent biotechnological applications-e.g. engineering synthetic formatotrophy and methylotrophy.IMPORTANCEC1 substrates continue to attract interest in biotechnology, as their use is both cost-effective and ultimately expected to mitigate the impact of greenhouse gas emissions. However, our current understanding of bacterial C1 metabolism remains relatively limited in species that cannot grow on (i.e., assimilate) these substrates. Pseudomonas putida, a model Gram-negative environmental bacterium, constitutes a prime example of this sort. The biochemical pathways active in response to methanol, formaldehyde, and formate have been largely overlooked-although the ability of P. putida to process C1 molecules has been previously alluded to in the literature. By using a systems-level strategy, this study bridges such knowledge gap through the identification and characterization of mechanisms underlying methanol, formaldehyde, and formate detoxification-including hitherto unknown enzymes that act on these substrates. The results reported herein both expand our understanding of microbial metabolism and lay a solid foundation for engineering efforts toward valorizing C1 feedstocks

Extracellular degradation of a polyurethane oligomer involving outer membrane vesicles and further insights on the degradation of 2,4-diaminotoluene in <i>Pseudomonas capeferrum</i> TDA1

The continuing reports of plastic pollution in various ecosystems highlight the threat posed by the ever-increasing consumption of synthetic polymers. Therefore, Pseudomonas capeferrum TDA1, a strain recently isolated from a plastic dump site, was examined further regarding its ability to degrade polyurethane (PU) compounds. The previously reported degradation pathway for 2,4-toluene diamine, a precursor and degradation intermediate of PU, could be confirmed by RNA-seq in this organism. In addition, different cell fractions of cells grown on a PU oligomer were tested for extracellular hydrolytic activity using a standard assay. Strikingly, purified outer membrane vesicles (OMV) of P. capeferrum TDA1 grown on a PU oligomer showed higher esterase activity than cell pellets. Hydrolases in the OMV fraction possibly involved in extracellular PU degradation were identified by mass spectrometry. On this basis, we propose a model for extracellular degradation of polyester-based PUs by P. capeferrum TDA1 involving the role of OMVs in synthetic polymer degradation