A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants.

BACKGROUND: Hydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits. RESULTS: The putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine. CONCLUSION: SrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism.

Enhanced Expression of Pullulanase in Bacillus subtilis by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination.

Pullulanase plays an important role as a starch hydrolysis enzyme in the production of bio-fuels and animal feed, and in the food industry. Compared to the methods currently used for pullulanase production, synthesis by Bacillus subtilis would be safer and easier. However, the current yield of pullulanase from B. subtilis is low to meet industrial requirements. Therefore, it is necessary to improve the yield of pullulanase by B. subtilis. In this study, we mined 10 highly active promoters from B. subtilis based on transcriptome and bioinformatic data. Individual promoters and combinations of promoters were used to improve the yield of pullulanase in B. subtilis BS001. Four recombinant strains with new promoters (Phag, PtufA, PsodA, and PfusA) had higher enzyme activity than the control (PamyE). The strain containing PsodA+fusA (163 U/mL) and the strain containing PsodA+fusA+amyE (336 U/mL) had the highest activity among the analyzed dual- and triple-promoter construct stains in shake flask, which were 2.29 and 4.73 times higher than that of the strain with PamyE, respectively. Moreover, the activity of the strain containing PsodA+fusA+amyE showed a maximum activity of 1,555 U/mL, which was 21.9 times higher than that of the flask-grown PamyE strain in a 50-liter fermenter. Our work showed that these four strong promoters mined from transcriptome data and their combinations could reliably increase the yield of pullulanase in quantities suitable for industrial applications.

Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal.

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that have an important role in plant development and adaptation to environmental changes. In this study, we identified and characterized a spermidine hydroxycinnamoyl transferase (SHT) from eggplant (Solanum melongena) and its wild relative S. richardii, designated as SmSHT and SrSHT, respectively. SmSHT was abundant in flowers and fruits, whereas the level of SrSHT was remarkably low in all tissues. Heat-shock/drought treatment stimulated the expression of SmSHT in both leaves and fruits, indicating its involvement in plant stress response. Both SHT polypeptides had extremely high identity with just five amino-acid substitutions. Recombinant SmSHT catalyzed the synthesis of mono-, bi- and tri- acylated polyamines. Using caffeoyl-CoA as the acyl donor, SmSHT preferred spermidine as the acyl acceptor. When spermidine was the acyl acceptor, the donor preference order for SmSHT was caffeoyl-CoA>feruloyl-CoA>ρ-coumaroyl-CoA. SrSHT exhibited the same substrate specificity as SmSHT, yet exhibited significantly higher catalytic activity than SmSHT. For example, under caffeoyl-CoA and spermidine, Kcat of SrSHT was 37.3% higher than SmSHT. Molecular modeling suggests that five amino-acid substitutions in SrSHT result in four alterations in their predicted 3D structures. In particular, the conserved Lys402 adjacent to the DFGWG motif, and Cys200 in the crossover loop in SmSHT were replaced by Glu and Ser in SrSHT. These substitutions may contribute to the enhanced activity in SrSHT. Our study provides a platform to generate HCAA rich fruits for eggplant and other solanaceous crops.