Introduction of alkali-labile units into lignin in transgenic plants by genetic engineering

Background Lignin is one of major components of plant secondary cell wall. In plant cell wall, it is synthesized via radical coupling of precursors such as p-coumaryl, coniferyl, and sinapyl alcohols. In early stage of the lignification, 8-O-4’, 8-8’ and 8-5’ dimers are thought to be synthesized mainly from the precursors in the wall. A gramnegative bacterium, Shingobium sp. strain SYK-6 (hereafter refer to as SYK-6) is able to catabolize a wide variety of phenolic compounds including the lignin precursors by its unique enzymatic system. One of catabolic enzymes, LigD, catalyzes oxidation at alpha (benzyl) position of 8-O-4’ dimers and forms carbonyl group at the position (Figure 1). This oxidation is the first step of catabolic pathway of 8-O-4’ dimers in SYK-6. When we express LigD polypeptide in the cell wall of transgenic plants, the oxidative dimers will be expected to be generated and then incorporated into lignin polymer. In some past studies, it has been shown that the presence of carbonyl groups at the alpha position of aryl propane units in lignin greatly speeds up the rate of cleavage of beta-aryl ether linkages during kraft pulping condition [1,2]. In order to contribute to efficient and sustainable production of kraft pulp and the other biomass-derived products such as bioethanol, we introduced the ligD gene into Arabidopsis and hybrid aspen and tried to generate transgenic plants whose lignin can be easy to remove from hollocellulose fraction under alkaline conditions. Method Because of codon usage is significantly different between genes in plants and SYK-6, we chemically synthesized open reading frame (ORF) of the ligD gene for improving its expression in the transgenic plants. After addition of nucleotide sequence for apoplast-targeting signal peptide to the synthesized ligD ORF, it was introduced into Arabidopsis thaliana, tobacco BY-2 and hybrid aspen under the control of cauliflower mosaic virus 35S promoter. LigD expression in the transgenic plants was monitored by Western blot analysis and enzymatic activity with crude extract prepared from each transgenic line. Preliminary analysis of lignin structure by 2D-NMR and nitrobenzene oxidation was also performed.