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For decades, plastic waste management has been one of the major ecological challenges of our society. Despite the introduction of biodegradable alternatives such as polylactic acid (PLA), their beneficial environmental impact is limited by the requirement of specific compost facility as biodegradation of PLA in natural environment occurs at a very slow rate. In this work, a plastic-degrading enzyme was utilized to facilitate degradation process. Genomic and proteomic tools were employed to identify a new biodegradable plastic-degrading enzyme from Cryptococcus nemorosus TBRC2959. The new enzyme, Cr14CLE, functions optimally under mild conditions with temperature range of 30 to 40 °C and suffers no significant loss of enzymatic activity at pH ranging from 6 to 8. In addition to PLA, Cr14CLE is capable to degrade other types of biodegradable plastic such as polybutylene succinate (PBS) and polybutylene adipate terephthalate (PBAT) as well as composite bioplastic. Applications of Cr14CLE have been demonstrated through the preparation of enzyme-coated PLA film and laminated PLA film with enzyme layer. PLA films prepared by both approaches exhibited capability to self-degrade in water. Key points: • Novel plastic-degrading enzyme (Cr14CLE) was identified and characterized. • Cr14CLE can degrade multiple types of biodegradable plastics under mild conditions. • Applications of Cr14CLE on self-degradable plastic were demonstrated. [ABSTRACT FROM AUTHOR]

The threat of global plastic waste accumulation has spurred the exploration of plastics derived from biological sources. A well-known example is polyester made of 1,3-propanediol (1,3-PDO). However, there is no known pathway to assimilate 1,3-PDO into the central carbon metabolism, posing a potential challenge to upcycling such plastic wastes. Here, we proposed that the 1,3-PDO assimilation pathway could pass through malonate semialdehyde (MSA) as an intermediate. Since MSA is a toxic aldehyde, β-alanine was chosen as a surrogate substrate in this study to construct the lower part of the proposed pathway. To this end, we successfully engineered E. coli MG1655 to assimilate β-alanine as the major carbon source. β-alanine could be easily converted into MSA using a β-alanine/pyruvate transaminase from Pseudomonas aeruginosa (PaBapt). However, the subsequent step to generate acetyl-CoA from MSA was unknown. After a series of phenotype screenings, adaptive laboratory evolution and transcriptomic analysis, two CoA-acylating MSA dehydrogenases from Vibrio natriegens (VnMmsD), were found to be able to complete the metabolic pathway. Optical density at 600 nm (OD600) of the resulting strain E. coli BA02 could reach 4.5 after 96 h. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsDs were expressed from a single plasmid to mitigate antibiotic stress. Second, a native 3-hydroxy acid dehydrogenase (EcYdfG) was disrupted to address the carbon loss to 3-hydroxypropionate (3-HP) production from MSA. OD600 of the best-performing strain E. coli BA07∆ could reach 6 within 24 h using 5 g/L β-alanine. The construction of E. coli BA07∆ lays a solid foundation to establishing a 1,3-PDO assimilation pathway. Keypoints: • This study demonstrates the implementation of a metabolic pathway to assimilate β-alanine as the major carbon source in E. coli MG1655. • Two V. natriegens CoA-acylating methyl malonate semialdehyde dehydrogenases were used to complete the pathway in E. coli BA02. • The construction of E. coli BA02 also revealed the plasmid fusion event between two plasmids with the same replication origin. [ABSTRACT FROM AUTHOR]