Secretory overproduction of a raw starch-degrading glucoamylase in Penicillium oxalicum using strong promoter and signal peptide
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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.
KeywordsInducible promoter Signal peptide Raw starch-degrading glucoamylase Penicillium oxalicum
JXF designed and supervised the research and was involved in the data analysis and the preparation of the manuscript. SZ cosupervised all the research and was involved in the analysis of all experimental data and the preparation of the manuscript. LW did the experiments of construction of deletion mutants, overexpressed strains, green fluorescence intensity measurement, and enzyme activity tests and was involved in the preparation of the manuscript. XXC was involved in the screening of candidate promoters and signal peptides in P. oxalicum. QPD was involved in data analysis. CXL performed bioinformatic analysis and primer design. All authors have read and approved the final manuscript.
This work was supported by the Guangxi BaGui Scholars Program Foundation (Grant No. 2011A001) and the Guangxi Natural Science Foundation (Grant No. 2012GXNSFGA060005) to JXF and the “One Hundred Person” Project of Guangxi to SZ.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
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