Evolution in a test tube
Lignin is the most abundant aromatic natural polymer on Earth and can be a key renewable source of high value chemicals, materials and fuel precursors. Millions of tonnes of lignin preparations are produced by the paper industry every year but lignin is currently considered a biowaste because of its inherent heterogeneity and recalcitrance to degradation. Finding optimized and environment friendly biological routes for lignin valorization have been the focus of intensive research. Indeed for most industrial process applications the conditions are so far different from those found in nature that organisms and their enzymes need to be modified to achieve the necessary properties. The development of laboratory engineering strategies has been followed in Lígia Martins Lab and their most recent results in collaboration with a group from ISEL/Técnico have just been published at ACS Catalysis.
ITQB NOVA researchers used directed evolution approaches, as an alternative to rationally designing modified proteins, on a bacterial Pseudomonas putida DyP-type peroxidase. After only three rounds of random mutagenesis and screening, the resulting variant enzyme showed 100 times more activity towards the oxidation of phenolic and aromatic amines, lignin model compounds and Kraft lignin itself, with an optimal pH shifted to alkaline values, resistance to hydrogen peroxide inhibition and enhanced production yields, providing clear advantages for application in lignocellulose bioprocesses. The obtained evolved variants were thoroughly characterized and the role of acquired mutations was unveiled from the catalytic, stability and structural viewpoints.
“The idea of “made-to-order” enzymes is both fascinating and challenging and it can serve a variety of purposes in different fields. This is possible through the application of directed evolution which mimics the natural evolution cycle in a laboratory setting, not an easy route but certainly the most effective and exciting approach to engineer enzymes”, said Lígia Martins, main author of the work. “We will continue to explore the potential of engineering enzymes for different purposes, and look forward to seeing the applications and the fundamental knowledge derived”.
ACS Catal. 2017, 7, 3454−3465 DOI 10.1021/acscatal.6b03331
Vânia Brissos, Diogo Tavares, Ana Catarina Sousa, Maria Paula Robalo and Lígia O. Martins