Annually, 400 Mt of plastics are produced of which roughly 40% is discarded within a year. Current plastic waste management approaches focus on applying physical, thermal, and chemical treatments... Show moreAnnually, 400 Mt of plastics are produced of which roughly 40% is discarded within a year. Current plastic waste management approaches focus on applying physical, thermal, and chemical treatments of plastic polymers. However, these methods have severe limitations leading to the loss of valuable materials and resources. Another major drawback is the rapid accumulation of plastics into the environment causing one of the biggest environmental threats of the twenty-first century. Therefore, to complement current plastic management approaches novel routes toward plastic degradation and upcycling need to be developed. Enzymatic degradation and conversion of plastics present a promising approach toward sustainable recycling of plastics and plastics building blocks. However, the quest for novel enzymes that efficiently operate in cost-effective, large-scale plastics degradation poses many challenges. To date, a wide range of experimental set-ups has been reported, in many cases lacking a detailed investigation of microbial species exhibiting plastics degrading properties as well as of their corresponding plastics degrading enzymes. The apparent lack of consistent approaches compromises the necessary discovery of a wide range of novel enzymes. In this review, we discuss prospects and possibilities for efficient enzymatic degradation, recycling, and upcycling of plastics, in correlation with their wide diversity and broad utilization. Current methods for the identification and optimization of plastics degrading enzymes are compared and discussed. We present a framework for a standardized workflow, allowing transparent discovery and optimization of novel enzymes for efficient and sustainable plastics degradation in the future. Show less
Bacterial biocatalysis constitutes a sustainable alternative for high-value chemicals production by enabling the utilization of renewable feedstocks. However, biobased production ofaromatic... Show moreBacterial biocatalysis constitutes a sustainable alternative for high-value chemicals production by enabling the utilization of renewable feedstocks. However, biobased production ofaromatic compounds and biopolymers requires a specialized microbial cell factory. Microbial hosts may experience cell toxicity caused by the solvent-like compounds that emerge as products, substrates or intermediates during the production process. Therefore, solvent tolerance is an essential trait for the microbial hosts used in biobased production of aromatic chemicals and biopolymers. The work described in this thesis focused on identifying and characterizing genes/gene clusters which are involved in conferring solvent tolerance trait in bacteria. Adaptability of P. putida S12 is dependent on the ability to cope with the high energy demand of solvent stress. The inherent metabolic flexibility of P. putida S12 has partly been developed through horizontally transferred traits, such as aromatic degradation pathways and solvent extrusion pumps. Show less
Kusumawardhani, H.; Furtwängler, B.; Blommestijn, M.; Kaltenytė, A.; Poel, J.J.A. van der; Kolk, J.; ... ; Winde, J.H. de 2020
Pseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds.... Show morePseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that P. putida S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating P. putida S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA, respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with the slvAT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as P. putida KT2440, Escherichia coli TG1, and E. coli BL21(DE3). Show less
Kusumawardhani, H.; Hosseini, R.; Winde, J.H. de 2018
The challenge of sustainably producing highly valuable chemical compounds requires specialized microbial cell factories because many of these compounds can be toxic to microbial hosts. Therefore,... Show moreThe challenge of sustainably producing highly valuable chemical compounds requires specialized microbial cell factories because many of these compounds can be toxic to microbial hosts. Therefore, solvent-tolerant bacteria are promising production hosts because of their intrinsic tolerance towards these compounds. Recent studies have helped to elucidate the molecular mechanisms involved in solvent tolerance. Advances in synthetic biological tools will enable further development of streamlined solvent-tolerant production hosts and the transfer of solvent-tolerant traits to established industrial strains. In this review, we outline challenges and opportunities to implement solvent tolerance in bacteria as a desired trait for industrial biotechnology. Show less
