Gene duplications have increased the diversity and specificity of enzymes used by beetle larvae to break down important wood components
Larvae of longhorn species mainly develop in woody tissue, which is difficult to digest. However, the larvae have special enzymes with which they break down various components of the plant cell wall. Researchers at the Max Planck Institute for Chemical Ecology in Jena have now examined a group of digestive enzymes that occur only in this family of beetles. They were able to restore a primordial enzyme that first appeared in the common ancestor of longhorned beetles. Horizontal gene transfer from the bacterium to the beetle, as well as old and new gene duplications, enabled the evolution of this family of digestive enzymes and enabled the longhorns to break down the major components of the plant cell wall, which make up the majority of their diet. .
Longhorn larvae play an important role in the forest ecosystem because they feed almost exclusively on the wood of dead trees and thus return nutrients to the natural cycle. Larvae of some longhorn species eat the wood of living trees or also develop in treated wood and thus can cause considerable damage. Wood is created by lignification, i.e. the incorporation of lignin into the plant cell wall. Its main components are the polysaccharides cellulose and xylan and the polymer lignin, which are difficult for most organisms to break down. Like leafhoppers, weevils and bark beetles, longhorns have enzymes to break down plant cell wall components. Some enzyme families are widespread in these beetle families, but GH5_2 enzymes have so far only been found in longhorn beetles.
A team of researchers from the Max Planck Institute for Chemical Ecology and the Great Lakes Forest Center in Canada therefore wondered what is so special about these enzymes and why they are only found in longhorns. “Some of our previous work has shown that in at least one subfamily of longhorn beetles, the so-called weaver bugs, members of the GH5_2 enzyme family have evolved to break down not only cellulose but also other polysaccharides, such as xylan and xyloglucan. We wanted to find out if these catalytic abilities are also limited to weaver beetles or if it is a kind of trademark of all longhorn beetles,” explains study leader Yannick Pauchet.
The researchers combined phylogenetic analyzes with the generation of large functional datasets. The result of testing a total of 113 GH5_2 enzymes from 25 species is a comprehensive analysis of the evolutionary history of these enzymes in longhorns. Scientists have discovered that their function, that is, which polysaccharides they can break down, is related to their relationship. All genes are caused by at least four gene duplications (gene duplications).
From the comparative studies, the researchers were able to derive the gene sequence of the original enzyme, which first appeared in the ancestor of today’s longhorned beetle after bacterial transmission. They successfully expressed it in cell cultures and tested its activity on a number of important plant cell wall polysaccharides. “These experiments provided important evidence that this ancestral enzyme, cellulase, not only degraded cellulose but was ‘promiscuous’, i.e. it also catalyzed the conversion of other substrates such as xylan and glucomannan. This ability could have been a prerequisite for the development of the substrate specificity of these enzymes after gene duplication,” says first author Na Ra Shin. A surprising result for the researchers was that they encountered an enzyme that prefers transglycosylation, i.e. the attachment of a sugar group, to hydrolysis, i.e. cleavage “Only extensive screening of enzyme activity, as we have done here, can enable the discovery of enzymes with such properties,” says Na Ra Shin.
Gene exchange between species
Research on the ability of insects to digest certain plant food components shows that horizontal gene transfer played a key role. “There is more and more evidence that the acquisition of new metabolic capacities through horizontal gene transfer has played an important role in the evolution of many living things,” says Yannick Pauchet with certainty.
A systematic approach, which in this work combines sequence discovery and functional characterization of a complete enzyme family in a range of beetle species, is also a powerful way to identify catalytically interesting enzymes for potential biotechnological applications. Of special interest here are enzymes that are able to perform the reverse reaction, i.e. sugar transfer and binding or transglycosylation instead of cleavage. “Some polysaccharides have the potential of therapeutic application. However, their synthesis is difficult and requires long-term chemical processes with low yields. Alternatively, glycosyltransferases can be used, but they are very unstable and require expensive donor substrates. The use of transglycosylation enzymes can circumvent all these drawbacks. With our approach, we discovered a natural transglycosidase. It’s a little sensation,” says Na Ra Shin happily.
It would be interesting for researchers to gain a more comprehensive understanding of the importance of these enzymes to longhorn biology. However, this is only possible with the help of mutants. “Until now, it has simply not been possible to generate mutants with altered enzyme activity in these insects because the beetles spend most of their lives hiding in woody tissue, their development time can be several years, and it is not easy to grow them in the laboratory. “, says Yannick Pauchet