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

Abstract

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. IMPORTANCE C1 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.

Synergistic investigation of natural and synthetic C1-trophic microorganisms to foster a circular carbon economy

“…Achieving an effective plug&play implantation of C1 assimilation in established cell factories will open the possibility of quickly testing it into different genetic backgrounds. This might be particularly important as different species might be innately more or less tolerant to high concentrations of C1 compounds 73 , 88 , 131 . As many polytrophic microorganisms have been dominating the biotech industry in the past, a wealth of information is available about their physiology and upscaling to industrial settings.…”

Automated in vivo enzyme engineering accelerates biocatalyst optimization

“…Relevant examples include cofactors such as pyrroloquinoline quinone (PQQ; redox coenzyme in dehydrogenases) or heme (prosthetic group for oxygen-carrying or electron transfer). PQQ is a common coenzyme for alcohol dehydrogenases in P. putida 143 or Methylobacterium extorquens 144 ; and while the heme biosynthetic pathway is present in E. coli 145 , other bacteria, such as the metal-reducing Shewanella oneidensis , have many more enzymes that require this prosthetic group 146 – 148 .…”