Figure 9

Impact of Acthi over-expression on CPC production in the HY strain. (A) PCR verification of pYG239/HY. Lanes 1, 2, and 3: genomic DNA of HY-Acthi(+)-1.2.3; lane 4: genomic DNA of HY; lane 5: positive control; M: DL2000 DNA Marker. The phleo gene was detected in the Acthi mutants. (B) Acthi expression levels in the HY-Acthi(+) transformants. The relative mRNA abundance levels were normalized against the actin gene levels. Error bars represent the standard deviations from three independent experiments. Significant differences in the Acthi expression levels in HY-Acthi(+) mutants compared with HY are indicated as “*”; p value confidence levels: “*” < 0.05, “**” < 0.01, and “***” < 0.001. The Acthi gene transcription levels in HY-Acthi(+)-1,2,3 were higher compared to those in the HY strain during fermentation. (C) Western blotting analysis of ActhiS expression in HY-Acthi(+) transformants. Fifteen micrograms of total protein isolated from fermented mycelia on day 7 was loaded for Western blotting analysis with anti-ActhiS antibodies. Actin was used as the loading control. The expression levels of ActhiS in HY-Acthi(+)-1,2,3 were higher compared to those in the HY strain during the fermentation. (D) Comparison of CPC production in the HY strain and in the mutants HY-Acthi(+)-1,HY-Acthi(+)-2 and HY-Acthi(+)-3 on fermentation day 7. Data were presented as the means ± SD (n = 3). The CPC production of the transformants was significantly improved over that of the HY stain.