Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling

The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy

In vivo and in vitro FMN prenylation and (de)carboxylase activation under aerobic conditions

Prenylated FMN (prFMN) is a newly discovered redox cofactor required for activity of the large family of reversible UbiD (de)carboxylases involved in biotransformation of aromatic, heteroaromatic, and unsaturated aliphatic acids (White et al., 2015). Despite the growing demand for decarboxylases in the pulp/paper industry and in forest biorefineries, the vast majority of UbiD-like decarboxylases remain uncharacterized. Functional characterization of the novel UbiD decarboxylases is hindered by the lack of prFMN generating system. prFMN cofactor is synthesized by the UbiX family of FMN prenyltransferases, which use reduced FMN as substrate under anaerobic conditions and dimethylallyl-monophosphate (DMAP) as the prenyl group donor. Here, we report the in vivo and in vitro biosynthesis of prFMN and UbiD activation under aerobic conditions. For in vivo biosynthesis, we used newly discovered UbiX proteins from Salmonella typhimurium and Klebsiella pneumonia, which activated ferulic acid UbiD decarboxylase Fdc1 from Aspergillus niger under aerobic conditions (0.5-1.5 U/mg). For in vitro biosynthesis of prFMN and UbiD activation, we established a one-pot enzyme cascade system that uses prenol, polyphosphate, formate, and riboflavin as starting substrates and (re)generates DMAP, ATP, FMN and NADH. The system contains 6 different enzymes: prenol kinase, polyphosphate kinase, formate dehydrogenase, FMN reductase, riboflavin kinase and FMN prenyltransferase. Under aerobic conditions, this system showed up to 80% conversion of FMN to prFMN and generated active Fdc1 decarboxylase (0.2-1 U/mg). Thus, both systems represent robust approaches for in vivo and in vitro prFMN biosynthesis and UbiD activation under aerobic conditions. The developed FMN prenylation systems will facilitate the exploration and biochemical characterization of UbiD-like decarboxylases and their applications in biocatalysis. Please click Additional Files below to see the full abstract

A novel C-terminal protein degron identified in bacterial aldehyde decarbonylases using directed enzyme evolution

Metabolic engineers have successfully synthesized alkanes, the bulk component of gasoline, using microbial cell factories as a sustainable alternative to petroleum-based fuels. Aldehyde decarbonylases (AD), enzymes which transform acyl aldehydes into alkanes, have been identified as the bottleneck in these alkane producing pathways. Previous studies demonstrated degradation of AD in E. coli cells via unknown molecular mechanism. Here, we present the discovery of a degradation tag (degron) in AD from Prochlorococcus marinus. AD variants were generated by random mutation using error-prone PCR, transferred into E. coli, and grown in chemostat culture with 2g/L hexanal to select for positive mutations. A short C-terminal sequence of AD from P. marinus was proven to be an intact degron by fusing to fluorescent proteins. Statistical analysis of C-terminal sequences of 371 non-redundant ADs from bacteria revealed a conserved sequence in this region, which was proven to be an effective degron. We also showed that ATP-dependent proteases clpAP and lon are responsible for the degradation of AD degron tagged protein. Furthermore, our results indicate that the AD degron caused 91.4% of green fluorescent protein (GFP) degradation when fused to its C-terminus, whereas its elimination in AD enhanced alkane production in vivo. Thus, our work demonstrated the presence of a protein degron tag in bacterial ADs, thereby facilitating further improvements in AD-based alkane production pathways. Please click Additional Files below to see the full abstract

Complete biosynthesis of adipic acid in Saccharomyces cerevisiae

Adipic acid is an important industrial chemical that is produced in significant quantities every year. However, conventional processes for its production are not sustainable due to a heavy dependence on petroleum derived feedstocks and emission of greenhouse gases. Biotechnological production of adipic acid in a yeast host is a sustainable alternative that can overcome these issues. While many heterologous pathways have been proposed to achieve this, significant progress has been made only using the muconic acid pathway which has been implemented by many research groups in both E. coli and S. cerevisiae. However, the in vivo conversion of muconic acid to adipic acid has not been reported. In this work, we describe the isolation of a novel enzyme: 2-enoate reductase that is capable of reducing the pi bond of an alpha unsaturated carboxylic acid such as muconic acid. We have characterized the substrate profile of these novel enzymes and have identified an oxygen tolerant enoate reductase that has significant potential for adipic acid production. This enzyme was tested for muconic acid activity in S. cerevisiae and was then expressed in a muconic acid producing yeast strain to construct a yeast host that is capable of complete biosynthesis of adipic acid using glucose as the only feedstock. To our knowledge, this is the first reported yeast strain that is capable of adipic acid biosynthesis using glucose as the only feedstock. We anticipate that adipic acid production can be improved further through metabolic engineering. Please click Additional Files below to see the full abstract

Enzymatic biotransformation of adipic acid to 6-aminocaproic acid and 1,6- hexamethylenediamine using engineered carboxylic acid reductases and aminotransferases

Biocatalytic reduction of carboxylic acids is gaining importance for the production of polymer precursors and different chemicals. Carboxylic acid reductases (CARs) reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Recently, we demonstrated that several bacterial CARs can reduce a broad range of bifunctional carboxylic acids containing amino group or second carboxylic group including adipic acid, which is a precursor for nylon-6-6 (Khusnutdinova et al., 2017). In this project, we demonstrate application of CARs and aminotransferases for further bioconversion of adipic acid to 6-aminocaproic acid and hexamethylenediamine, two other important precursors for nylon synthesis. Based on the crystal structure of the adenylating domain of the CAR enzyme MCH22995 from Mycobacterium chelonae, we generated a structural model of the CAR enzyme MAB4714 from M. abscessus, which is active toward adipic acid. Aiming at improving MAB4714 activity toward 6-aminocaproic acid, we used structure-based protein engineering and generated 16 MAB4714 mutant proteins. Screening of 16 purified MAB4714 variants against 6-aminocaproic acid,identified one protein, which was 10 times more active than the wild-type protein. We also identified several bacterial aminotransferases producing 6-aminocaproic acid from adipic acid in combination with CARs. Further optimization of reaction conditions and application of cofactor regeneration systems resulted in efficient biotransformation of adipic acid to 6-aminocaproic acid (88% conversion) and further to 1,6-hexamethylenediamine (78% conversion). Please click Additional Files below to see the full abstract

Moderately thermostable GH1 β-glucosidases from hyperacidophilic archaeon Cuniculiplasma divulgatum S5

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilised in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterised. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5–6.0 (CIB12) and 4.5–5.5 (CIB13) with optimal temperatures at 50 °C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma