Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species

Indexación: Scopus.The use of renewable waste feedstocks is an environment-friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by-products. Glycerol (1,2,3-propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by-product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole-cell biocatalysts with both wild-type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value-added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost-effective substrate. The unique genetic and metabolic features of glycerol-grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate – and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.1340

Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level

Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells

Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens

Bioremediation 3.0: Engineering pollutant-removing bacteria in the times of systemic biology

Elimination or mitigation of the toxic effects of chemical waste released to the environment by industrial and urban activities relies largely on the catalytic activities of microorganisms—specifically bacteria. Given their capacity to evolve rapidly, they have the biochemical power to tackle a large number of molecules mobilized from their geological repositories through human action (e.g., hydrocarbons, heavy metals) or generated through chemical synthesis (e.g., xenobiotic compounds). Whereas naturally occurring microbes already have considerable ability to remove many environmental pollutants with no external intervention, the onset of genetic engineering in the 1980s allowed the possibility of rational design of bacteria to catabolize specific compounds, which could eventually be released into the environment as bioremediation agents. The complexity of this endeavour and the lack of fundamental knowledge nonetheless led to the virtual abandonment of such a recombinant DNA-based bioremediation only a decade later. In a twist of events, the last few years have witnessed the emergence of new systemic fields (including systems and synthetic biology, and metabolic engineering) that allow revisiting the same environmental pollution challenges through fresh and far more powerful approaches. The focus on contaminated sites and chemicals has been broadened by the phenomenal problems of anthropogenic emissions of greenhouse gases and the accumulation of plastic waste on a global scale. In this article, we analyze how contemporary systemic biology is helping to take the design of bioremediation agents back to the core of environmental biotechnology. We inspect a number of recent strategies for catabolic pathway construction and optimization and we bring them together by proposing an engineering workflow.PD is the holder of the Marie Sklodowska-Curie grant No. 704410 (FUTURE). The work in Authors' Laboratory was funded by the CAMBIOS Project of the Spanish Ministry of Economy and CompetitivenessRTC-2014-1777-3 (MINECO), HELIOS Project of the Spanish Ministry of Economy and Competitiveness BIO 2015-66960-C3-2-R (MINECO/FEDER) and the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), and Raft4Biotech (720776) contracts of the European Union. JD is supported also by the Czech Ministry of Education (LQ1605, LO1214, LM2015055), and Czech Grant Agency (GA16-06096S).Peer Reviewe

Running headline: Engineering PHA production in P. putida

28 p.-6 fig.-1 tab.This article is dedicated to the memory of our colleague, Dr. Shane Kenny, who made significant contributions towards developing bioprocesses for sustainable PHA production. The scientific community is deeply saddened by his untimely demise in 2020.BACKGROUND: Growing environmental concern sparked 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 towards 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.PURPOSE AND SCOPE: The surge of synthetic biology and metabolic engineering approaches in recent years now offers the possibility of exploiting the untapped potential of Pseudomonas-based cell factories for the production of tailored PHAs. In this article, we provide an overview of the metabolic and regulatory circuits that rule PHA accumulation in P. putida, and we discuss approaches leading to the biosynthesis of novel polymers (e.g. PHAs including non-biological chemical elements in their structures).CONCLUSION: The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. We conclude the review by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and we propose strategies towards programmable polymer biosynthesis from alternative substrates in engineered P. putida strains.This work was supported by grants from The Novo Nordisk Foundation (NNF10CC1016517 and NNF18OC0034818—LiFe), the European Union’s Horizon2020 Research and Innovation Programme under grant agreement No. 814418 (SinFonia) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B) to P.I.N. M.M. acknowledges the support of the Novo Nordisk Foundation. We also acknowledge support from the Community of Madrid (P2018/NMT4389) and the Spanish Ministry of Science, Innovation, and Universities (BIO2017-83448-R) to M.A.P.Peer reviewe

Bio-upcycling of even and uneven medium-chain-length diols and dicarboxylates to polyhydroxyalkanoates using engineered Pseudomonas putida

Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations. Graphical Abstract: (Figure presented.)</p