As a frequent inhabitant of sites polluted with toxic chemicals, the soil bacterium and plant-root colonizer Pseudomonas putida can tolerate high levels of endogenous and exogenous oxidative stress. Yet, the ultimate reason of such phenotypic property remains largely unknown. To shed light on this question, metabolic network-wide routes for NADPH generation—the metabolic currency that fuels redox-stress quenching mechanisms—were inspected when P. putida KT2440 was challenged with a sub-lethal H2O2 dose as a proxy of oxidative conditions. 13C-tracer experiments, metabolomics, and flux analysis, together with the assessment of physiological parameters and measurement of enzymatic activities, revealed a substantial flux reconfiguration in oxidative environments. In particular, periplasmic glucose processing was rerouted to cytoplasmic oxidation, and the cyclic operation of the pentose phosphate pathway led to significant NADPH-forming fluxes, exceeding biosynthetic demands by ~50%. The resulting NADPH surplus, in turn, fueled the glutathione system for H2O2 reduction. These properties not only account for the tolerance of P. putida to environmental insults—some of which end up in the formation of reactive oxygen species—but they also highlight the value of this bacterial host as a platform for environmental bioremediation and metabolic engineering.

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Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress.

Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point toward renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs. The surge of synthetic biology and metabolic engineering approaches in recent years offers the possibility of exploiting the untapped potential of Pseudomonas cell factories for the production of tailored PHAs. In this article, an overview of the metabolic and regulatory circuits that rule PHA accumulation in Pseudomonas putida is provided, and approaches leading to the biosynthesis of novel polymers (e.g., PHAs including nonbiological chemical elements in their structures) are discussed. The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. The review is concluded by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and strategies toward programmable polymer biosynthesis from alternative substrates in engineered P. putida strains are proposed.

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

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Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways.

A navigation guide of synthetic biology tools for Pseudomonas putida.

Biobased furans, such as furfural, 5-hydroxymethylfurfural, and 2,5-furandicarboxylic acid, are recognized as the top-value platform chemicals in the valorization of biomass to chemicals, fuels, and materials. Biocatalysis has emerged as a promising technique in the chemical and pharmaceutical industries because of exquisite selectivity, mild reaction conditions, environmental friendliness, etc. In this comprehensive review, we summarize the recent advances in (chemo)biocatalytic conversion of biobased furans, based on the reaction types including oxidation, reduction, C–C and C–N bonds formation, esterification, and amidation. Particularly, the biocatalysts and their catalytic mechanisms/pathways are critically discussed to allow the rational design and construction of efficient enzymes/multienzyme systems/chemobiocatalytic systems. Besides, various biocatalyst engineering and reaction engineering strategies are highlighted to intensify the processes. Future research directions are proposed to expand the chemical space of furans as well as to improve the commercial acceptance of biocatalytic processes. 

(Chemo)biocatalytic Upgrading of Biobased Furanic Platforms to Chemicals, Fuels, and Materials: A Comprehensive Review

Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. KEY POINTS: • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.

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Industrial biotechnology of Pseudomonas putida: advances and prospects

Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties.

Anna Weimer

Papers

Industrial biotechnology of Pseudomonas putida: advances and prospects

Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties.