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 ß-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 ß-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous ß-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.

Characterization and engineering of branched short-chain dicarboxylate metabolism in Pseudomonas reveals resistance to fungal 2-hydroxyparaconate.

In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building blocks for plastics urges for developing microbial upcycling strategies to provide sustainable end-of-life solutions. Furthermore, many BSCD exhibit anti-bacterial properties or exert immunomodulatory effects in macrophages, indicating a medical relevance for this group of molecules. For both of these applications, a detailed understanding of the microbial metabolism of these compounds is essential. In this study, the metabolic pathway of BSCD degradation from Pseudomonas aeruginosa PAO1 was studied in detail by heterologously transferring it to Pseudomonas putida. Heterologous expression of the PA0878-0886 itaconate metabolism gene cluster enabled P. putida KT2440 to metabolize itaconate, (S)- and (R)-methylsuccinate, (S)-citramalate, and mesaconate. The functions of the so far uncharacterized genes PA0879 and PA0881 were revealed and proven to extend the substrate range of the core degradation pathway. Furthermore, the uncharacterized gene PA0880 was discovered to encode a 2-hydroxyparaconate (2-HP) lactonase that catalyzes the cleavage of the itaconate derivative 2-HP to itatartarate. Interestingly, 2-HP was found to inhibit growth of the engineered P. putida on itaconate. All in all, this study extends the substrate range of P. putida to include BSCD for bio-upcycling of high-performance polymers, and also identifies 2-HP as promising candidate for anti-microbial applications.

The metabolic potential of plastics as biotechnological carbon sources - Review and targets for the future.

The plastic crisis requires drastic measures, especially for the plastics' end-of-life. Mixed plastic fractions are currently difficult to recycle, but microbial metabolism might open new pathways. With new technologies for degradation of plastics to oligo- and monomers, these carbon sources can be used in biotechnology for the upcycling of plastic waste to valuable products, such as bioplastics and biosurfactants. We briefly summarize well-known monomer degradation pathways and computed their theoretical yields for industrially interesting products. With this information in hand, we calculated replacement scenarios of existing fossil-based synthesis routes for the same products. Thereby, we highlight fossil-based products for which plastic monomers might be attractive alternative carbon sources. Notably, not the highest yield of product on substrate of the biochemical route, but rather the (in-)efficiency of the petrochemical routes (i.e., carbon, energy use) determines the potential of biochemical plastic upcycling. Our results might serve as a guide for future metabolic engineering efforts towards a sustainable plastic economy.

Global Regulator of Rubber Degradation in Gordonia polyisoprenivorans VH2: Identification and Involvement in the Regulation Network.

A cAMP receptor protein (CRPVH2) was detected as a global regulator in Gordonia polyisoprenivorans VH2 and was proposed to participate in the network regulating poly(cis-1,4-isoprene) degradation as a novel key regulator. CRPVH2 shares a sequence identity of 79% with GlxR, a well-studied global regulator of Corynebacterium glutamicum Furthermore, CRPVH2 and GlxR have a common oligomerization state and similar binding motifs, and thus most likely have similar functions as global regulators. Size exclusion chromatography of purified CRPVH2 confirmed the existence as a homodimer with a native molecular weight of 44.1 kDa in the presence of cAMP. CRPVH2 bound to the TGTGAN6TCACT motif within the 131-bp intergenic region of divergently oriented lcp1VH2 and lcpRVH2, encoding a latex clearing protein and its putative repressor, respectively. DNase I footprinting assays revealed the exact operator size of CRPVH2 in the intergenic region (25 bp), which partly overlapped with the proposed promoters of lcpRVH2 and lcp1VH2 Our findings indicate that CRPVH2 represses the expression of lcpRVH2 while simultaneously directly or indirectly activating the expression of lcp1VH2 by binding the competing promoter regions. Furthermore, binding of CRPVH2 to upstream regions of additional putative enzymes of poly(cis-1,4-isoprene) degradation was verified in vitro. In silico analyses predicted 206 CRPVH2 binding sites comprising 244 genes associated with several functional categories, including carbon and peptide metabolism, stress response, etc. The gene expression regulation of several subordinated regulators substantiated the function of CRPVH2 as a global regulator. Moreover, we anticipate that the novel lcpR regulation mechanism by CRPs is widespread in other rubber-degrading actinomycetes.IMPORTANCE In order to develop efficient microbial recycling strategies for rubber waste materials, it is required that we understand the degradation pathway of the polymer and how it is regulated. However, only little is known about the transcriptional regulation of the rubber degradation pathway, which seems to be upregulated in the presence of the polymer. We identified a novel key regulator of rubber degradation (CRPVH2) that regulates several parts of the pathway in the potent rubber-degrader G. polyisoprenivorans VH2. Furthermore, we provide evidence for a widespread involvement of CRP regulators in the degradation of rubber in various other rubber-degrading actinomycetes. Thus, these novel insights into the regulation of rubber degradation are essential for developing efficient microbial degradation strategies for rubber waste materials by this group of actinomycetes.

Impact of additives of commercial rubber compounds on the microbial and enzymatic degradation of poly(cis-1,4-isoprene).

Much fundamental research has already been performed to understand the mechanism of microbial rubber degradation. Due to the increasing amount of rubber waste, biotechnical methods to degrade that particular waste are strongly needed. The present study evaluates whether a microbial or an enzymatic process is more suitable for efficient biodegradation, due to less sensitivity towards rubber additives. Therefore we investigated the impact of 15 different frequently used rubber additives on cells of the potent rubber degrader Gordonia polyisoprenivorans VH2 and the enzyme Lcp1VH2. For this, cells were grown on poly(cis-1,4-isoprene) in presence of these rubber additives. Furthermore, the effect of those additives on the enzymatic cleavage of poly(cis-1,4-isoprene) by Lcp1VH2 was determined by in vitro studies. It was observed that additives, used to accelerate the vulcanization process, like N-cyclohexyl-2-benzothiazolesulfenamide and zinc-bis(N,N-dibenzyl-dithiocarbamate), are diminishing the growth of the microorganism depending on their concentration-higher toxicity with increasing concentration. In contrast, sulfur prevents cell growth, but does not affect Lcp1VH2. Stearic acid and paraffin wax were found to be consumed by G. polyisoprenivorans VH2. Plasticizers mainly prevent growth, but do not interfere with the enzyme activity. This study identified antioxidants as the most interfering group of additives for microbial and enzymatic rubber degradation. It was found that the in vitro degradation by Lcp1VH2 is much more resistant and less sensitive towards the investigated rubber additives, when compared to the in vivo approach. Therefore, an enzymatic process might be a promising method to enhance rubber degradation.

Halopseudomonas species: Cultivation and molecular genetic tools.

The Halopseudomonas species, formerly classified as Pseudomonas pertucinogena lineage, form a unique phylogenetic branch within the Pseudomonads. Most strains have recently been isolated from challenging habitats including oil- or metal-polluted sites, deep sea, and intertidal zones, suggesting innate resilience to physical and chemical stresses. Despite their comparably small genomes, these bacteria synthesise several biomolecules with biotechnological potential and a role in the degradation of anthropogenic pollutants has been suggested for some Halopseudomonads. Until now, these bacteria are not readily amenable to existing cultivation and cloning methods. We addressed these limitations by selecting four Halopseudomonas strains of particular interest, namely H. aestusnigri, H. bauzanensis, H. litoralis, and H. oceani to establish microbiological and molecular genetic methods. We found that C4 -C10 dicarboxylic acids serve as viable carbon sources in both complex and mineral salt cultivation media. We also developed plasmid DNA transfer protocols and assessed vectors with different origins of replication and promoters inducible with isopropyl-ß-d-thiogalactopyranoside, l-arabinose, and salicylate. Furthermore, we have demonstrated the simultaneous genomic integration of expression cassettes into one and two attTn7 integration sites. Our results provide a valuable toolbox for constructing robust chassis strains and highlight the biotechnological potential of Halopseudomonas strains.

Biodegradation of poly(ester-urethane) coatings by Halopseudomonas formosensis.

Impranil® DLN-SD is a poly(ester-urethane) (PEU) that is widely used as coating material for textiles to fine-tune and improve their properties. Since coatings increase the complexity of such plastic materials, they can pose a hindrance for sustainable end-of-life solutions of plastics using enzymes or microorganisms. In this study, we isolated Halopseudomonas formosensis FZJ due to its ability to grow on Impranil DLN-SD and other PEUs as sole carbon sources. The isolated strain was exceptionally thermotolerant as it could degrade Impranil DLN-SD at up to 50°C. We identified several putative extracellular hydrolases of which the polyester hydrolase Hfor_PE-H showed substrate degradation of Impranil DLN-SD and thus was purified and characterized in detail. Hfor_PE-H showed moderate temperature stability (Tm = 53.9°C) and exhibited activity towards Impranil DLN-SD as well as polyethylene terephthalate. Moreover, we revealed the enzymatic release of monomers from Impranil DLN-SD by Hfor_PE-H using GC-ToF-MS and could decipher the associated metabolic pathways in H. formosensis FZJ. Overall, this study provides detailed insights into the microbial and enzymatic degradation of PEU coatings, thereby deepening our understanding of microbial coating degradation in both contained and natural environments. Moreover, the study highlights the relevance of the genus Halopseudomonas and especially the novel isolate and its enzymes for future bio-upcycling processes of coated plastic materials.