The management and recycling of plastic waste is a challenging global issue. Polyethylene terephthalate (PET), one of the most widely used synthetic plastics, can be hydrolyzed by a series of enzymes. However, upcycling the resulting monomers is also a problem. In this study, we designed a co-cultivation system, in which PET degradation was coupled with polyhydroxybutyrate (PHB) production. First, PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase. The engineered PETase-producing Y. lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl) terephthalate (BHET) and PET powder into the monomers terephthalate (TPA) and ethylene glycol (EG). Simultaneously, a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phbCAB operon from Ralstonia eutropha, which encodes enzymes for the biosynthesis of PHB. The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step. During this process, 5.16 g/L BHET was hydrolyzed in 12 h, and 3.66 wt% PHB (3.54 g/L cell dry weight) accumulated in 54 h. A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h. Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase, this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.

Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.

Summary: With the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR‐based gene expression toolkit of Y. lipolytica . By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR‐based gene expression toolkit developed in this study will facilitate the genetic engineering of Y . lipolytica .

Polyolefin plastics, such as polyethylene (PE) and polystyrene (PS), are the most widely used synthetic plastics in our daily life. However, the chemical structure of polyolefin plastics is composed of carbon-carbon (C-C) bonds, which is extremely stable and makes polyolefin plastics recalcitrant to degradation. The growing accumulation of plastic waste has caused serious environmental pollution and has become a global environmental concern. In this study, we isolated a unique Raoultella sp. DY2415 strain from petroleum-contaminated soil that can degrade PE and PS film. After 60 d of incubation with strain DY2415, the weight of the UV-irradiated PE (UVPE) film and PS film decreased by 8% and 2%, respectively. Apparent microbial colonization and holes on the surface of the films were observed by scanning electron microscopy (SEM). Furthermore, the Fourier transform infrared spectrometer (FTIR) results showed that new oxygen-containing functional groups such as -OH and -CO were introduced into the polyolefin molecular structure. Potential enzymes that may be involved in the biodegradation of polyolefin plastics were analyzed. These results demonstrate that Raoultella sp. DY2415 has the ability to degrade polyolefin plastics and provide a basis for further investigating the biodegradation mechanism.