ABSTRACT
Raw starch-degrading enzymes (RSDEs) are capable of directly degrading raw starch granules below the gelatinization temperature of starch, which may significantly reduce the cost of starch-based biorefining. However, low yields of natural RSDEs from filamentous fungi limit their industrial application. In this study, transcriptomic and secretomic profiling was employed to screen strongest promoters and signal peptides for use in overexpression of a RSDE gene in Penicillium oxalicum. Top five strong promoters and three signal peptides were detected. Using a green fluorescent protein (GFP) as the reporter, the inducible promoter pPoxEgCel5B of an endoglucanase gene PoxEgCel5B and the signal peptide spPoxGA15A of a raw starch-degrading glucoamylase PoxGA15A were respectively identified as driving the highest GFP production in P. oxalicum. PoxGA15A-overexpressed P. oxalicum strain OXPoxGA15A, which was constructed based on both pPoxEgCel5B and spPoxGA15A, produced significantly higher amounts of recombinant PoxGA15A than the parental strain ∆PoxKu70. Furthermore, crude enzyme from the OXPoxGA15A strain exhibited high activities towards raw starch from cassava, potato, and uncooked soluble starch. Specifically, raw cassava starch-degrading enzyme activity reached 241.6 U/mL in the OXPoxGA15A, which was 3.4-fold higher than that of the ∆PoxKu70. This work provides a feasible method for hyperproduction of RSDEs in P. oxalicum.
Subject(s)
Glucan 1,4-alpha-Glucosidase/genetics , Penicillium/genetics , Promoter Regions, Genetic/genetics , Protein Sorting Signals/genetics , Starch/genetics , Fermentation/genetics , Fungi/genetics , Manihot/genetics , Recombinant Proteins/genetics , Solanum tuberosum/genetics , Starch/metabolism , TemperatureABSTRACT
Orychophragines A-C (1-3), three new alkaloids with an novel 2-piperazinone-fused 2,4-dioxohexahydro-1,3,5-triazine skeleton, were isolated from the seeds of Orychophragmus violaceus. Their structures were established on the basis of spectroscopic analysis and X-ray crystallographic analysis. Orychophragines A (1) exhibited remarkable cytotoxicity against HepG2, A549, Hela, and HCT-116 cells with IC50 values of 7.73, 10.79, 11.91, and 9.93 µM, respectively. Orychophragines C (3) showed moderate 60Co γ radiation protection activity in HUVEC cells. A plausible biosynthetic pathway for 1-3 was proposed.
