Jie Li

In plants, the diverse enzymes known as BAHD acyltransferases use acyl coenzyme A (acyl-CoA) as their substrate and fulfill crucial biological functions. In this study, 94 NtBAHD genes were identified in the tobacco ( Nicotiana tabacum ) genome. These genes displayed considerable diversity in their physicochemical properties, phylogenetic relationships, gene structures, and conserved motifs. Chlorogenic acid (CGA), which is the predominant polyphenol found in tobacco leaves, is mainly synthesised via the hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) pathway. Four HQT genes ( NtHQT1 – NtHQT4 ) were identified in the BAHD gene family of N. tabacum . Of these, NtHQT1 and NtHQT2 were highly expressed in leaves, while NtHQT3 and NtHQT4 were root-specific. Following heterologous expression in vitro , three of the four HQT proteins (NtHQT2, NtHQT3, and NtHQT4) were obtained in soluble form; however, only NtHQT2 and NtHQT3 exhibited enzymatic activity. Downregulation of NtHQT1 , NtHQT2 , NtHQT3 , and NtHQT4 significantly reduced both total polyphenol and chlorogenic acid (CGA) content, whereas the levels of rutin, scopoletin, and lignin remained unchanged. Furthermore, the expression of NtHQT genes was differentially induced under abiotic and biotic stresses, including drought, cold, and Phytophthora nicotianae infection. In contrast, their responses to various phytohormones exhibited distinct expression profiles. These results suggest that whereas in vitro assays identified NtHQT2 and NtHQT3 as the catalytically active isoforms, all four NtHQT genes contribute to chlorogenic acid (CGA) biosynthesis and environmental adaptation. Trial registration This study does not involve clinical trials or human participants and, as such, does not require clinical trial registration.

As a kind of Amaryllidaceae alkaloid which is accumulated in the species of Lycoris plants, lycorine has a range of physiological effects. The biosynthesis pathway of lycorine has been partly revealed, but the transport and accumulation mechanisms of lycorine have rarely been studied. In this study, an ATP-binding cassette (ABC) transporter from Lycoris aurea (L’Hér) Herb., namely LaABCB11, was cloned and functionally characterized. Heterologous expression showed that LaABCB11 transported lycorine in an outward direction, increased the tolerance of yeast cells to lycorine, and caused a lower lycorine accumulation in transformants than control or mutant in yeast. LaABCB11 is associated with the plasma membrane, and in situ hybridization indicated that LaABCB11 was mainly expressed in the phloem of leaves and bulbs, as well as in the cortical cells of roots. These findings suggest that LaABCB11 functions as a lycorine transport and it might be related to the translocation and accumulation of lycorine from the leaves and bulbs to the roots.

p-Coumaric acid is a commercially available phenolcarboxylic acid with a great number of important applications in the nutraceutical, pharmaceutical, material and chemical industries. p-Coumaric acid has been biosynthesized in some engineered microbes, but the potential of the plant CYP450-involved biosynthetic route has not investigated in Escherichia coli. In the present study, a novel trans-cinnamic acid 4-hydroxylase (C4H) encoding the LauC4H gene was isolated from Lycoris aurea (L’ Hér.) Herb via rapid amplification of cDNA ends. Then, N-terminal 28 amino acids of LauC4H were characterized, for the subcellular localization, at the endoplasmic reticulum membrane in protoplasts of Arabidopsis thaliana. In E. coli, LauC4H without the N-terminal membrane anchor region was functionally expressed when fused with the redox partner of A. thaliana cytochrome P450 enzyme (CYP450), and was verified to catalyze the trans-cinnamic acid to p-coumaric acid transformation by whole-cell bioconversion, HPLC detection and LC-MS analysis as well. Further, with phenylalanine ammonia-lyase 1 of A. thaliana, p-coumaric acid was de novo biosynthesized from glucose as the sole carbon source via the phenylalanine route in the recombinant E. coli cells. By regulating the level of intracellular NADPH, the production of p-coumaric acid was dramatically improved by 9.18-fold, and achieved with a titer of 156.09 μM in shake flasks. The recombinant cells harboring functional LauC4H afforded a promising chassis for biological production of p-coumaric acid, even other derivatives, via a plant CYP450-involved pathway.