Poly(ethylene terephthalate) (PET) is one of the world’s most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensis and the ICCG variant of leaf-branch compost cutinase (LCC) were codon-optimized and expressed in Escherichia coli BL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCC so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCC. The resulting four different LCC variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2 g/L. Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200 g/L recycled PET in a well-controlled, stirred-tank reactor. The results will help develop an economical and scalable biorecycling process toward a circular PET economy.

The United States produces more than 10 million tons of waste oils and fats each year. This paper aims to establish a new biomanufacturing platform that convert waste oils or fats into a series of value-added products. Our research employs the oleaginous yeast Yarrowia lipolytica as a case study for citrate production from waste oils. First, we conducted the CFD simulation of the bioreactor system and identified that the extracellular mixing and mass transfer is the first limiting factor of an oil fermentation process due to the insolubility of oil in water. Based on the CFD simulation results, bioreactor design and operating conditions were optimized and successfully enhanced oil uptake and bioconversion in fed-batch fermentation experiments. After that, we investigated the impacts of cell morphology on oil uptake, intracellular lipid accumulation, and citrate formation by overexpressing and deleting the MHY1 gene in the wild type Y. lipolytica. Fairly good correlations were achieved between cell morphology and productivities of biomass, lipid, and citrate. Finally, fermentation kinetics with both glucose and oil substrates were compared and the oil fermentation process was carefully evaluated. Our research results suggest that waste oils or fats can be economical feedstocks for biomanufacturing of many high-value products.