Synthetic enzyme reduces lignin content

Public domain image. Source: Glazer, A. W., and Nikaido, H. (1995). Microbial Biotechnology: fundamentals of applied microbiology. San Francisco: W. H. Freeman, p. 340. ISBN 0-71672608-4

Highlighted article: Kewwi Zhang, Mohammad-Wadud Bhuiya, Jorge Rencoret Pazo, Yuchen Miao, Hoon Kim, John Ralph, and Chang-Jun Liu (2012) An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis. Plant Cell Preview.

Zhang et al. reduce lignin content by introducing an artificial enzyme to the cell wall biosynthesis pathway. This is the first time synthetic biology has been used to change cell wall structure, which is usually modified by changing the expression of endogenous enzymes or introducing a protein from another organism. In fact at the moment, synthetic biology is not a common method of manipulating any plant pathway.

Relevant background

public domain image, courtesy of Chino

Lignin is one of three components of secondary cell walls. It is the part which makes extracting sugar from the cell wall, for example for second generation biofuel production, difficult.

Lignin is made up of three monolignols: coniferyl, sinapyl, and p-coumaryl.

They are synthesised in the cytosol and transported to the cell wall. At the cell wall, the monolignols are oxidised, causing their phenol group to become radicalised. The phenoxy radicals polymerise to form the lignin macromolecule.

The Liu lab had the idea of preventing monolignol oxidation by methylation of the phenol group so that the phenoxy radicals were prevented from forming. Their first attempt was to synthesise a selection of monolignol 4-O-methyltransferases (MOMTs). The artificial MOMTS were fusions of two naturally occurring enzymes: lignin biosynthesis pathway methyltransferase COMT, which does not have any 4-O-methyltransferase activity; and fairy fan enzyme isoeugenol O-methyltransferase, which catalyzes 4-O-methylation of isoeugenol and eugenol, but doesn’t affect monolignols. Although several of these artificial enzymes were able to 4-O-methylate monolignols as expected in vitro, they had no activity in vivo.

Results

Zhang et al. used MOMT3, a promising enzyme from their earlier work, as a starting point. They studied the crystal structure of the enzyme bound to a coniferyl alcohol substrate and S-adenosyl homocysteine (SAH), the product of methyl donation by S-adenosyl Met (SAM). Having identified 20 amino acids that formed the active site and were in contact or close proximity to the coniferyl alcohol, the team used mutagenesis to change each of the chosen 20 in turn. They identified three which when substituted caused the new enzyme, now called MOMT4, to have increased activity – roughly 200-fold more than the original IEMT wt enzyme.

When tested in vitro, MOMT 4 successfully carried out 4-O-methylation of uncoupled phenol on monolignols, which prevented the monolignols from becoming free radicals and blocking oligomerisation as hoped. Monolignols that avoided 4-O-methylation were able to polymerise as normal. In vivo, the scientists confined expression of MOMT4 to sites of lignin biosynthesis by expressing it under the bean PAL2 promotor. Lignin content was reduced and sugar release from cellulose treated cell walls was increased by 22% compared to wildtype Arabidopsis.

MOMT4 in this system was quite specific, methylating only coniferyl and sinapyl alcohols and their aldehydes. All other lignin precursers, including p-coumaryl and caffeyl alcohols, were unaffected.

Other than extra wall-bound phonolics, which Zhang et al. detected using LC-MS, and slightly early senescence compared to the wiltdype, the plants grew and developed normally. No phenotypic difference was visible in the size or shape of the plant cells or the plant as a whole. There was no difference in cellulose content and no noticeable difference in global gene expression.

Teaching resources:

NREL have an activity on how genes can be changed to reduce lignin and hemicellulose and increase cellulose content in secondary cell walls.



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