Pachymic acid (PA), a triterpenoid from Poria cocos, has various pharmacological effects, including anti-inflammatory, anti-cancer, anti-aging, and insulin-like properties. PA has gained considerable research attention, but the mechanism of its anti-cancer effects remains unclear. In this study, pyruvate kinase M2 (PKM2) was discovered as a PA target via the drug affinity responsive target stability. Molecular docking and enzyme assay revealed that PA is a competing activator of PKM2, and mimics the natural activator, fructose-1,6-bisphosphate. PKM2 activation should augment the flux of glycolysis. However, decreased glucose uptake and lactate production after PA treatment was observed in SK-BR-3 breast carcinoma cells, indicating a blockage or downregulation of glycolysis. The potential of previously reported triterpenoids in blocking hexokinase II (HK2) activity inspired us to investigate the inhibition effect of PA on HK2 activity. Molecular docking and enzyme assay confirmed that PA was an inhibitor of HK2, with an IC 50 of 5.01 µM. The possible consequences of glycometabolic regulation by PA, such as dissociation of HK2 from the mitochondria, release of mitochondrial cytochrome (Cyt) c, depletion of ATP, and generation of reactive oxygen species, were further validated. Furthermore, the details of the possible linkage of targeting PKM2 and HK2 with previously reported actions of PA were discussed. The results of our study provided valuable information on the anti-cancer mechanisms of PA.

The experiments of elicitation and in situ adsorption were conducted in shake flasks and then tested in a modified bubble column bioreactor for enhancing the productions of three active metabolites in Tripterygium wilfordii Hook. f., triptolide, wilforgine and wilforine. Methyl jasmonate was screened out as the elicitor and the non-ionic polymeric ion-exchange resin of Amberlite(®) XAD-7 was used for in situ product removal and protecting the alkaloids from degradation in the medium. In shake flask experiments, 3.55-fold, 49.11-fold, and 10.40-fold of triptolide, wilforgine, and wilforine, respectively, could be recovered from the medium and XAD-7 resin by elicitation and in situ product removal, compared with the control. The modified 10 L bubble column bioreactor had similar productions of the three active metabolites but needed a further optimization of parameters for better growth of adventitious roots.

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A filamentous actinomycete, designated strain ZX01(T), was isolated from forest soil around Kanas Lake of China. A polyphasic taxonomic study was carried out to establish the status of strain ZX01(T). Chemical and morphological properties of the isolate were similar to those of species of the genus Streptomyces. Analysis of the almost complete 16S rRNA gene sequence placed strain ZX01(T) in the genus Streptomyces where it formed a distinct phyletic line with recognized species of this genus. The strain exhibited the highest sequence similarities to Streptomyces lavendofoliae NBRC 12882(T) (99.1%), S. luridus NBRC 12793(T) (99.0%), S. lavendulocolor NBRC 12881(T) (99.0%), S. gobitricini NBRC 15419(T) (99.0%), and S. roseolilacinus NBRC 12815(T) (98.9%). Low DNA-DNA relatedness values of 54.0, 50.0, 60.0, 66.7, and 50.4%, respectively, were found between strain ZX01(T) and corresponding strains above. A number of phenotypic properties also enabled the isolate to be differentiated from related species of the genus Streptomyces. Therefore, it is proposed that strain ZX01(T) should be classified as the type strain of a novel species in the genus Streptomyces, Streptomyces kanasensis sp. nov. The type strain is ZX01(T) (= CGMCC 4893(T) =JCM 30232(T)).

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In order to solve the shortage of natural Tripterygium wilfordii Hook. f. plant resource for the production of the important secondary metabolites triptolide and wilforine, hairy roots were induced from its root calli by Agrobacterium rhizogenes. Induced hairy roots not only could be maintained and grown well in hormone-free half-strength Murashige and Skoog medium but also could produce sufficient amounts of both triptolide and wilforine. Although hairy roots produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots, they could grow fast and could be a suitable system for producing both secondary metabolites compared with other tissues. Addition of 50 micrometer methyl jasmonate (MeJA) could slightly affect hairy root growth, but dramatically stimulated the production of both triptolide and wilforine, whereas 50 micrometer salicylic acid had no apparent effect on hairy root growth with slightly stimulatory effects on the production of both secondary metabolites. Addition of precursor nicotinic acid, isoleucine, or aspartic acid at the concentration of 500 micrometer had varying effects on hairy root growth, but none of them had stimulatory effects on triptolide production, and only the former two had slightly beneficial effects on wilforine production. The majority of triptolide produced was secreted into the medium, whereas most of the produced wilforine was retained inside of hairy roots. Our studies provide a promising way to produce triptolide and wilforine in T. wilfordii hairy root cultures combined with MeJA treatment.

TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids. Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.

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