The story of a serendipitous microbial Alley-oop
"An alley-oop in basketball is an offensive play in which one player throws the ball near the basket to a teammate who jumps, catches the ball in mid air and puts it in the hoop before touching the ground." - Wikipedia
Two years ago, the media spotlight shone upon an unassuming bacterial species called Ideonella sakaiensis because it lived in landfill sites but more importantly, because it was found to degrade and metabolise plastic. This ability was the result of the evolution of two enzymes that degraded PET into terephthalic acid and ethylene glycol. Without realising it, these plastic-eating bacteria had just thrown the perfect ball to within an inch of the hoop.
A transatlantic research team took one of the enzymes, the PETase and started mutating the active site in order to understand how it evolved. While evaluating the activity of the mutants, they found that one of the enzymes had improved activity. In other words, they took the ball from the bacteria and dunked it straight through the hoop.
“What actually turned out was we improved the enzyme, which was a bit of a shock,” said Prof John McGeehan, at the University of Portsmouth, UK, who led the research. “It’s great and a real finding.”
The team used the X-ray Crystallography beamline at the UK's synchrotron, Diamond Light Source, to determine the precise structure of the enzyme with the hope of putting it to use in the future, an endeavour which is not without challenges. McGeehan is however optimistic,
“You are always up against the fact that oil is cheap, so virgin PET is cheap,” said McGeehan. “It is so easy for manufacturers to generate more of that stuff, rather than even try to recycle. But I believe there is a public driver here: perception is changing so much that companies are starting to look at how they can properly recycle these.”
Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.
Characterization and engineering of a plastic-degrading aromatic polyesterase
Harry P. Austin, Mark D. Allen, Bryon S. Donohoe, Nicholas A. Rorrer, Fiona L. Kearns, Rodrigo L. Silveira, Benjamin C. Pollard, Graham Dominick, Ramona Duman, Kamel El Omari, Vitaliy Mykhaylyk, Armin Wagner, William E. Michener, Antonella Amore, Munir S. Skaf, Michael F. Crowley, Alan W. Thorne, Christopher W. Johnson, H. Lee Woodcock, John E. McGeehan and Gregg T. Beckham
PNAS April 17, 2018. 201718804; published ahead of print April 17, 2018. https://doi.org/10.1073/pnas.1718804115