26 11       4052.89 11       *Coefficients of determination for the regressions at 90% confidence level; SS: Sum of Squares; DF: Degrees of Freedom; MS: Mean Square. **F(5,6) at 90% confidence level = 3.11. Cephamycin C production was affected differently for lysine combined with the remaining four compounds. The resulting response surfaces of experimental designs using lysine and TPX-0005 in vitro alpha-aminoadipic acid

(Figure 4A) and lysine and 1,3-diaminopropane (Figure 4B) showed curves and parameters of the same order of magnitude, thereby providing comparable production values. The adjusted mathematical models provide the highest cephamycin C concentrations of approximately find more 126 and 140 mg l-1 when 0.6 g l-1 of alpha-aminoadipic acid and 5.3 g l-1 of lysine and 5.2 g l-1 of 1,3-diaminopropane and 7.0 g l-1 of lysine were added, respectively. In culture media containing MK-2206 research buy just lysine, a production of about 120 mg l-1 was obtained, but only at high amino acid concentrations (14.6 g l-1) (Figure 2).

It should be remarked that alpha-aminoadipic acid has a strong impact on cephamycin C production even when added at concentrations nine times lower than those of 1,3-diaminopropane. This is probably due to its being a direct precursor of the beta-lactam antibiotic molecule [20, 21, 33]. On the other hand, 1,3-diaminopropane acts indirectly on beta-lactam antibiotic biosynthesis at the genetic and transcriptional levels [32, 43]. Leitão et al. [32] showed that this diamine increases the concentration of lysine-6-aminotransferase and P6C dehydrogenase, which are enzymes responsible for alpha-aminoadipic acid formation. This complex mechanism may support the need for adding larger amounts of 1,3-diaminopropane to produce the same effect as that obtained with alpha-aminoadipic acid at lower concentrations, which is in line

with the results obtained in this study. These data and those found in the literature clearly demonstrate, albeit through different methods, that lysine conversion to alpha-aminoadipic acid is a limiting step to cephamycin C biosynthesis. For this reason, adding alpha-aminoadipic acid or 1,3-diaminopropane, though at different concentration levels, was equally effective to overcoming this bottleneck. Fitted response surfaces for cultivations in culture media containing lysine combined with cadaverine indicate that this diamine only exerts influence PAK5 on antibiotic production when lysine is added at low concentrations. When the amino acid concentration was increased, the effect of adding diamine gradually waned. It has been suggested that intracellular accumulation of cadaverine may regulate the lysine catabolic pathway through a feedback control mechanism. In this manner, the lysine that would be decarboxylated to form cadaverine is spared, thus increasing lysine supply for cephamycin biosynthesis via the alpha-aminoadipate pathway. The fitted model shows that this behavior only happens at low lysine concentrations.