Physalis peruviana L. ( P. peruviana ) is an edible medicinal plant rich in bioactive phenolics. This study aimed to establish a hairy root (HR) culture of P. peruviana as a potential source for the synthesis of natural compounds. HRs were induced in P. peruviana using different Agrobacterium rhizogenes strains (R1601, C58C1, A4, and K599). Notably, K599 did not induce HR formation, whereas R1601, C58C1, and A4 yielded transformation frequencies of 57.78, 65.14, and 72.31%, respectively. Secondary metabolite production and antioxidant capacity were further examined in HRs induced using C58C1, R1601, and A4. It was found that A. rhizogenes R1601 induced the greatest increase (44% compared to that observed in the non-transformed culture). The methanolic extract of HRs induced by A. rhizogenes R1601 exhibited strong antioxidant capacity, with IC 50 values of 1.41 mg DE/mL and 2.33 mg DE/mL for 2,2-diphenyl-1-picrylhydrazyl and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), respectively. The HR culture showed higher production of phenolic compounds and higher antioxidant capacity than the non-transformed cultures. Ultra-performance liquid chromatography time-of-flight tandem mass spectrometry was used to identify eight alkaloids, phenolics, and glycoside compounds. A. rhizogenes R1601 is emerging as a possible strain for the mass production of HR and bioactive phenolic compounds in P. peruviana .
Di Liu,Linbo Gou,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl ( S )-4-chloro-3-hydroxybutyrate (( S )-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency ( k cat / K m ) for COBE was 51.36 s –1 ·mM –1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.
Li Jingxin,Zhang Yuxiao,Wang Xing,Walk Seth T.,Wang Gejiao
Antimony (Sb)-resistant bacteria have potential applications in the remediation of Sb-contaminated sites. However, the effect of Sb(III) exposure on whole-cell metabolic change has not been studied. Herein, we combined untargeted metabolomics with a previous proteomics dataset and confirmatory gene transcription analysis to identify metabolic responses to Sb(III) exposure in Agrobacterium tumefaciens GW4. Dynamic changes in metabolism between control and Sb(III)-exposed groups were clearly shown. KEGG pathway analysis suggested that with Sb(III) exposure: (1) the branching pathway of gluconeogenesis is down-regulated, resulting in the up-regulation of pentose phosphate pathway to provide precursors of anabolism and NADPH; (2) glycerophospholipid and arachidonic acid metabolisms are down-regulated, resulting in more acetyl-CoA entry into the TCA cycle and increased capacity to produce energy and macromolecular synthesis; (3) nucleotide and fatty acid synthesis pathways are all increased perhaps to protect cells from DNA and lipid peroxidation; (4) nicotinate metabolism increases which likely leads to increased production of co-enzymes (e.g., NAD+ and NADP+) for the maintenance of cellular redox and Sb(III) oxidation. Expectedly, the total NADP+/NADPH content, total glutathione, and reduced glutathione contents were all increased after Sb(III) exposure in strain GW4, which contribute to maintaining the reduced state of the cytoplasm. Our results provide novel information regarding global bacterial responses to Sb(III) exposure from a single gene level to the entire metabolome and provide specific hypotheses regarding the metabolic change to be addressed in future research.
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