Figure 8

Effects of the Acthi gene on precursor amino acids and TPP-dependent enzymes in WT, Acthi(−) and Acthi(+). (A) Relative quantification of intracellular precursor amino acids (cysteine, valine and L-α-AAA) for CPC synthesis in A. chrysogenum mutants. Details of the sample preparation and GC/MS analysis for amino acids are described in the Methods. The concentrations of cysteine, valine and L-α-AAA showed similar trends in the Acthi mutants. (B) RT-qPCR analyses assessing the expression of TPP-dependent enzymes (PDC, TK and ALS) in A. chrysogenum mutants on fermentation day 7. The relative mRNA abundance levels were standardized against the actin gene levels. Error bars represent standard deviations from three independent experiments. Significant differences in the expression of TPP-dependent enzymes (PDC, TK and ALS) in Acthi(−) and Acthi(+) compared with WT are indicated as “*”; p value confidence levels: “*” < 0.05, “**” < 0.01, and “***” < 0.001. TK appeared to be most sensitive to the Acthi mutants. The PDC gene expression levels in Acthi(+) and Acthi(−) were not obviously significantly different. The ALS expression levels were increased by over 2-fold in Acthi(+) and showed a significant decline in Acthi(−).