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New Component of Lignin Biosynthesis Pathway Suggests Possible Biofuel Strategy

NEW YORK (GenomeWeb News) – An international team of researchers led by Claire Halpin at the University of Dundee in the UK and Wout Boerjan from the Flanders Institute for Biotechnology in Belgium reported in Science today that they have uncovered a previously unknown component of the lignin biosynthetic pathway. They narrowed in on caffeoyl shikimate esterase in part through a co-expression analysis of other genes involved in the process.

Lignin, along with polysaccharides like cellulose and hemicellulose, is a main component of plant cell walls. To make biofuels from plants, cellulose has to be converted to glucose for fermentation. But lignin gums up that process. It binds cellulose, forming a complex that allows plants to grow tall, though it impedes the breakdown of those plants for fuel.

Feedstocks with lower levels of lignin would make it easier to process cellulose for fuel as well as make that step less energy-hungry and, the researchers noted, cheaper. Arabidopsis thaliana plants with mutant versions of the caffeoyl shikimate esterase enzyme that the researchers found contained less lignin than wild type plants did, and the lignin that was present was beset by structural changes.

"This [finding] provides an alternative pathway for altering lignin in plants and has the potential to greatly increase the efficiency of energy crop conversion for biofuels," said Sally Benson, director of Stanford University's Global Climate and Energy Project, which partially funded the study, in a statement

To search for enzymes involved in lignification, the researchers teased out just which genes are expressed at the same time as genes encoding proteins known to make up the lignin biosynthetic pathway. By drawing on three different co-expression analysis methods — the Arabidopsis Co-expression Tool, CressExpress, and a high-resolution root spatiotemporal expression dataset — the researchers homed in on 13 co-expressed genes, three of which had no known role in the lignin pathway. Just one — caffeoyl shikimate esterase, or CSE — was confirmed as being expressed with lignin genes in a number of lignin mutants.

Halpin, Boerjan, and their colleagues developed two A. thaliana CSE mutants, a knockdown mutant and a knockout mutant, both of which had weak CSE transcript levels, as measured by qRT-PCR. While the knockdown mutant plants appeared to develop normally, the researchers noted that the knockout mutant plants were smaller and lighter. The knockouts also had collapsed vessel elements, which the researchers said were typical of plants with weak secondary cell walls.

Additionally, the mutants were made up of a smaller fraction by weight of cell wall polymers as compared to wild-type plants as well as other compositional shifts. The knockdown plants exhibited milder changes than the knockouts did, the researchers noted.

The researchers also sought to pinpoint where in the lignin biosynthetic pathway CSE was active. Based on liquid-chromatography mass spectrometry analysis of phenolics isolated from stem extracts from the mutant plants, they found that levels of the G and S lignin subunits were reduced. Meanwhile, NMR analysis indicated that levels of H subunit were increased by about 30 fold in the lignin of mutant plants. This indicated to the researchers that CSE has its effect in the biosynthesis pathway after the pathway for the G and S subunits diverged from that of the H subunit.

Further, the CSE mutant plants contained an abundance of a number of compounds that the researchers suspected might be the substrate upon which CSE acted. Indeed, caffeoyl shikimate was present at high levels in the plants, and upon the addition of recombinant CSE, it was nearly fully hydrolyzed into caffeic acid.

"We therefore suggest that caffeoyl shikimate is a substrate for CSE in vivo," the researchers wrote. "As caffeoyl shikimate is an accepted intermediate in lignin biosynthesis, this places CSE in the lignin biosynthetic pathway."

Mutant CSE plants have lower lignin levels and, as Halpin, Boerjan, and their colleagues pointed out, they might produce more sugars upon saccharification. And indeed, they found that to be the case when they compared mutant and wild-type plants.

Additionally, they found that a number of plants — including the biofuel feedstocks of poplar, eucalyptus, and switchgrass — contain CSE orthologs. "The characterization of CSE in other species will reveal how widely the revision of the lignin biosynthesis pathway we propose here applies and whether CSE could be a generally useful target for reducing cell wall recalcitrance to digestion or industrial processing in biomass crops," the investigators added.

Those feedstock plants could be screened for CSE mutants.

The researchers also noted that they have filed a patent pertaining to using CSE to engineer lignin in plants.