MIXed plastics biodegradation and UPcycling using microbial communities: EU Horizon 2020 project MIX-UP started January 2020.

This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese-European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits.

Engineering adipic acid metabolism in Pseudomonas putida.

Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant ß-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h-1, reaching a final biomass yield of 0.27 ± 0.00 gCDW gadipate-1. In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts.

The Inflection Point Hypothesis: The Relationship between the Temperature Dependence of Enzyme-Catalyzed Reaction Rates and Microbial Growth Rates.

The temperature dependence of biological rates at different scales (from individual enzymes to isolated organisms to ecosystem processes such as soil respiration and photosynthesis) is the subject of much historical and contemporary research. The precise relationship between the temperature dependence of enzyme rates and those at larger scales is not well understood. We have developed macromolecular rate theory (MMRT) to describe the temperature dependence of biological processes at all scales. Here we formalize the scaling relationship by investigating MMRT both at the molecular scale (constituent enzymes) and for growth of the parent organism. We demonstrate that the inflection point (Tinf) for the temperature dependence of individual metabolic enzymes coincides with the optimal growth temperature for the parent organism, and we rationalize this concordance in terms of the necessity for linearly correlated rates for metabolic enzymes over fluctuating environmental temperatures to maintain homeostasis. Indeed, Tinf is likely to be under strong selection pressure to maintain coordinated rates across environmental temperature ranges. At temperatures at which rates become uncorrelated, we postulate a regulatory catastrophe and organism growth rates precipitously decline at temperatures where this occurs. We show that the curvature in the plots of the natural log of the rate versus temperature for individual enzymes determines the curvature for the metabolic process overall and the curvature for the temperature dependence of the growth of the organism. We have called this "the inflection point hypothesis", and this hypothesis suggests many avenues for future investigation, including avenues for engineering the thermal tolerance of organisms.

Redundancy in putrescine catabolism in solvent tolerant Pseudomonas putida S12.

Pseudomonas putida S12 is a promising platform organism for the biological production of substituted aromatic compounds due to its extreme tolerance towards toxic chemicals. Solvent or aromatic stress tolerance may be due to membrane modifications and efflux pumps; however in general, polyamines have also been implicated in stressed cells. Previous transcriptomics results of P. putida strains producing an aromatic compound, or being exposed to the solvent toluene, indicated differentially expressed genes involved in polyamine transport and metabolism. Therefore, the metabolism of the polyamine, putrescine was investigated in P. putida S12, as no putrescine degradation pathways have been described for this strain. Via transcriptome analysis various, often redundant, putrescine-induced genes were identified as being potentially involved in putrescine catabolism via oxidative deamination and transamination. A series of knockout mutants were constructed in which up to six of these genes were sequentially deleted, and although putrescine degradation was affected in some of these mutants, complete elimination of putrescine degradation in P. putida S12 was not achieved. Evidence was found for the presence of an alternative pathway for putrescine degradation involving γ-glutamylation. The occurrence of multiple putrescine degradation routes in the solvent-tolerant P. putida S12 is indicative of the importance of controlling polyamine homeostasis, as well as of the high metabolic flexibility exhibited by this microorganism.

Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement.

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which--a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273--was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells.

Isolation and genetic characterization of an improved benzene-tolerant mutant of Pseudomonas putida S12.

Pseudomonas putida S12.49, a mutant stain of P. putida S12 that tolerates up to 20 mM benzene, was obtained by evolutionary selection. The genetic basis for the strongly enhanced benzene tolerance was investigated by proteome and transcriptome analysis. Indications were found that the highly benzene-tolerant phenotype is the resultant of multi-level systemic changes. The solvent extrusion pump SrpABC was constitutively expressed in P. putida S12.49, which could be attributed to the disruption of the srpS regulator gene by the indigenous mutator element ISS12. The occurrence of this and two additional transposition events was in good agreement with the increased transcriptional activity of transposase-encoding genes in strain S12.49. These observations suggested that transposition events are an important force driving the generation of the genetic diversity apparently required to obtain highly solvent-tolerant phenotypes. In addition, various expression responses relating to energy generation indicated system changes that accommodated the energy demand associated with the high-level expression of the proton-driven solvent extrusion pump. The relatively modest effect of a respiratory chain uncoupler on benzene tolerance in P. putida S12.49 indicated the involvement of an alternative, non-respiratory mechanism to maintain the proton gradient.

TrgI, toluene repressed gene I, a novel gene involved in toluene-tolerance in Pseudomonas putida S12.

Pseudomonas putida S12 is well known for its remarkable solvent tolerance. Transcriptomics analysis of this bacterium grown in toluene-containing chemostats revealed the differential expression of 253 genes. As expected, the genes encoding one of the major solvent tolerance mechanisms, the solvent efflux pump SrpABC and its regulatory genes srpRS were heavily up-regulated. The increased energy demand brought about by toluene stress was also reflected in transcriptional changes: genes involved in sugar storage were down-regulated whereas genes involved in energy generation such as isocitrate dehydrogenase and NADH dehydrogenases, were up-regulated in the presence of toluene. Several flagella-related genes were up-regulated and a large group of transport genes were down-regulated. In addition, a novel Pseudomonas-specific gene was identified to be involved in toluene tolerance of P. putida S12. This toluene-repressed gene, trgI, was heavily down-regulated immediately upon toluene exposure in batch cultures. The relationship of trgI with solvent tolerance was confirmed by the increased resistance to toluene shock and toluene induced lysis of trgI knock-out mutants. We propose that down-regulation of trgI plays a role in the first line of defence against solvents.

Transcriptome analysis of a phenol-producing Pseudomonas putida S12 construct: genetic and physiological basis for improved production.

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis.

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies.

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291(T)), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291(T). Pseudomonas monteilii (DSM14164) clustered well with P. putida strains.

Optimization of the solvent-tolerant Pseudomonas putida S12 as host for the production of p-coumarate from glucose.

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite L: -tyrosine. Efficient production was hampered by product degradation, limited cellular L: -tyrosine availability, and formation of the by-product cinnamate via L: -phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled L: -phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l(-1)) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.

Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose.

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.

Properties of a trichlorodibenzo-p-dioxin-dechlorinating mixed culture with a Dehalococcoides as putative dechlorinating species.

An anaerobic mixed culture enriched over 16 transfers (1/10) from Saale river sediment reductively dehalogenated 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) to di- and monochlorinated congeners in the presence of pyruvate (or lactate) and fumarate as cosubstrates. Besides TrCDD, tetrachloroethene and 1,2,3-trichlorobenzene were dechlorinated. Dioxin dehalogenation was sensitive to pasteurization, but was not remarkably influenced by inhibitors of methanogens, sulfate-reducing bacteria or Gram-positive bacteria. The rate of 1,3-dichlorodibenzo-p-dioxin formation increased with rising initial concentrations of 1,2,4-TrCDD (1-250 microM) from 0.05 to 5.4 micromol l(-1) day(-1). Two isolates, belonging to Sulfurospirillum and Trichococcus, did not show reductive dehalogenation. 16S rDNA-targeted methods further revealed the presence of Acetobacterium, Desulfitobacterium, Desulfuromonas and Dehalococcoides. Nested polymerase chain reaction (PCR) indicated the presence of Dehalococcoides in highest most probable number (MPN) dilutions that were positive for dioxin dechlorination.