Collective Total Synthesis of Mavacuran Alkaloids through Intermolecular 1,4-Addition of an Organolithium Reagent.

We report a synthetic endeavor towards the highly strained pentacyclic caged framework of the mavacuran alkaloids which culminated with the concise total synthesis of C-fluorocurine, C-profluorocurine, C-mavacurine, normavacurine, 16-epi-pleiocarpamine and taberdivarine H. We designed a strategy involving late-stage construction of the D ring by Michael addition of a vinylic nucleophile to a 2-indolyl acrylate moiety. While the intramolecular Michael addition did not succeed, we were able to perform a diastereoselective unusual intermolecular 1,4-addition of a functionalized vinyl lithium reagent to a readily accessible Michael acceptor with the assistance of the piperidine nitrogen atom through the formation of a complex as suggested by DFT computations. Final cyclization was achieved by nucleophilic substitution to form an ammonium intermediate. The first total syntheses of C-profluorocurine and C-fluorocurine were finalized by the dihydroxylation of C-mavacurine and a pinacol rearrangement, respectively.

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.

Towards bio-upcycling of polyethylene terephthalate.

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films are hydrolyzed by a thermostable polyester hydrolase yielding highly pure terephthalate and ethylene glycol. The obtained hydrolysate is used directly as a feedstock for a terephthalate-degrading Pseudomonas umsongensis GO16, also evolved to efficiently metabolize ethylene glycol, to produce PHA. The strain is further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which are used as monomers for the chemo-catalytic synthesis of bio-PU. In short, a novel value-chain for PET upcycling is shown that circumvents the costly purification of PET monomers, adding technological flexibility to the global challenge of end-of-life management of plastics.

Enzymatic recycling of thermoplastic polyurethanes: Synergistic effect of an esterase and an amidase and recovery of building blocks.

Biological recycling of polyurethanes (PU) is a huge challenge to take up in order to reduce a large part of the environmental pollution from these materials. However, enzymatic depolymerization of PU still needs to be improved to propose valuable and green solutions. The present study aims to identify efficient PU degrading enzymes among a collection of 50 hydrolases. Screenings based on model molecules were performed leading to the selection of an efficient amidase (E4143) able to hydrolyze the urethane bond of a low molar mass molecule and an esterase (E3576) able to hydrolyze a waterborne polyester polyurethane dispersion. Degradation activities of the amidase, the esterase and a mix of these enzymes were then evaluated on four thermoplastic polyurethanes (TPU) specifically designed for this assay. The highest degradation was obtained on a polycaprolactone polyol-based polyurethane with weight loss of 33% after 51 days measured for the esterase. Deep cracks on the polymer surface observed by scanning electron microscopy and the presence of oligomers on the remaining TPU detected by size exclusion chromatography evidenced the polymer degradation. Mixing both enzymes led to an increased amount of urethane bonds hydrolysis of the polymer. 6-hydroxycaproic acid and 4,4'-methylene dianiline were recovered after depolymerization as hydrolysis products. Such building blocks could get a second life with the synthesis of new macromolecular architectures.

Metabolic and transcriptomic adaptation of Lactococcus lactis subsp. lactis Biovar diacetylactis in response to autoacidification and temperature downshift in skim milk.

For the first time, a combined genome-wide transcriptome and metabolic analysis was performed with a dairy Lactococcus lactis subsp. lactis biovar diacetylactis strain under dynamic conditions similar to the conditions encountered during the cheese-making process. A culture was grown in skim milk in an anaerobic environment without pH regulation and with a controlled temperature downshift. Fermentation kinetics, as well as central metabolism enzyme activities, were determined throughout the culture. Based on the enzymatic analysis, a type of glycolytic control was postulated, which was shared by most of the enzymes during the growth phase; in particular, the phosphofructokinase and some enzymes of the phosphoglycerate pathway during the postacidification phase were implicated. These conclusions were reinforced by whole-genome transcriptomic data. First, limited enzyme activities relative to the carbon flux were measured for most of the glycolytic enzymes; second, transcripts and enzyme activities exhibited similar changes during the culture; and third, genes involved in alternative metabolic pathways derived from some glycolytic metabolites were induced just upstream of the postulated glycolytic bottlenecks, as a consequence of accumulation of these metabolites. Other transcriptional responses to autoacidification and a decrease in temperature were induced at the end of the growth phase and were partially maintained during the stationary phase. If specific responses to acid and cold stresses were identified, this exhaustive analysis also enabled induction of unexpected pathways to be shown.