Escherichia coli (Migula, 1895) Castellani & Chalmers, 1919

Zhang, Chi, Chen, Xinlu, Crandall-Stotler, Barbara, Qian, Ping, Köllner, Tobias G., Guo, Hong & Chen, Feng, 2019, Biosynthesis of methyl (E) - cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase, Phytochemistry 164, pp. 50-59 : 53

2.3. Biochemical characterization of CsSABATHs expressed in E. coli

Gene expression analysis ( Fig. 3 ) suggested that CsSABATH2, CsSABATH4 and CsSABATH6 are the most probable candidates as CAMT. To validate this prediction, we characterized the proteins encoded by all six CsSABATH genes using a biochemical approach. Full-length cDNA for each of the six CsSABATH genes was cloned by RT-PCR into a protein expression vector without any tag and expressed in E. coli to produce recombinant proteins. Each of the recombinant CsSABATH enzymes was assayed with (E)-cinnamic acid and an additional nine carboxyl acids: indole-3-acetic acid, gibberellin A3, salicylic acid, jasmonic acid, anthranilic acid, nicotinic acid, benzoic acid, p -coumaric acid and abscisic acid ( Table 1 ). Among the six CsSABATHs, CsSABATH6 was highly active using (E)-cinnamic acid as substrate. It also showed activity with benzoic acid and p -coumaric acid, which was about 32% and 18% of the activity with (E)-cinnamic acid. CsSABATH4 also showed some activity with (E)-cinnamic acid, but the activity was very low. Instead, CsSABATH4 was most active with salicylic acid as substrate. Its activity with (E)-cinnamic acid was only about 6% of that with salicylic acid. CsSABATH6 and CsSABATH4 were renamed CsCAMT (GenBank accession no. MK673137) and CsSAMT (GeneBank accession no. MK673138), respectively. CsSABATH1, CsSABATH2, CsSABATH3 and CsSABATH5 did not show activity with any of the substrates tested.

Figures

Fig. 3. Expression of six CsSABATHs genes in sterile thalli measured by semiquantitative RT-PCR. Same amount of cDNA was used in each reaction. This experiment was repeated three times with similar results. “M” indicates molecular marker.