Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser–His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.
Widespread
utilization of polyethylene terephthalate (PET) has
caused a variety of environmental and health problems; thus, the enzymatic
degradation of PET can be a promising solution. Although PETase from Ideonalla sakaiensis (IsPETase)
has been reported to have the highest PET degradation activity under
mild conditions of all PET-degrading enzymes reported to date, its
low thermal stability limits its ability for efficient and practical
enzymatic degradation of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy
using several IsPETase variants that were screened
for high thermal stability to improve PET degradation activity. In
particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7
connecting loop and extended subsite IIc, had a T
m value that was increased by 8.81 °C and PET degradation
activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications
were further verified through structure determination of the variants,
and high thermal stability was further confirmed by a heat-inactivation
experiment. The proposed strategy and developed variants represent
an important advancement for achieving the complete biodegradation
of PET under mild conditions.
Poly(lactate-co-glycolate) (PLGA) is a widely used biodegradable and biocompatible synthetic polymer. Here we report one-step fermentative production of PLGA in engineered Escherichia coli harboring an evolved polyhydroxyalkanoate (PHA) synthase that polymerizes D-lactyl-CoA and glycolyl-CoA into PLGA. Introduction of the Dahms pathway enables production of glycolate from xylose. Deletion of ptsG enables simultaneous utilization of glucose and xylose. An evolved propionyl-CoA transferase converts D-lactate and glycolate to D-lactyl-CoA and glycolyl-CoA, respectively. Deletion of adhE, frdB, pflB and poxB prevents by-product formation. We also demonstrate modulation of the monomer fractions in PLGA by overexpressing ldhA and deleting dld to increase the proportion of D-lactate or by deleting aceB, glcB, glcD, glcE, glcF and glcG to increase the proportion of glycolate. Incorporation of 2-hydroxybutyrate is prevented by deleting ilvA or feeding strains with L-isoleucine. The utility of our approach for generating diverse forms of PLGA is shown by the production of copolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate or 2-hydroxyisovalerate.
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