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(3) Because the starting precursor of acarbose derived from the pentose phosphate pathway, and NADPH was formed along with the pentose phosphate pathway, the cofactor (NADPH) regulation was predicted to affect acarbose production. (4) The expression of the acarbose cluster genes was different in different cultivation media (Schwientek et al., 2013); thus, the overexpression of the acb cluster genes would contribute to acarbose production. In silico simulation of acarbose production To increase the production of acarbose, three types of efforts were made. In model iYLW1028, the effect of additional amino acids on cell growth and acarbose production was simulated (Figure ?(Figure7).7). The results show that the addition of amino acids had a good effect on both cell growth and the acarbose production rate. The addition of arginine had the most significantly impact on acarbose production, followed by histidine. And compared to the control, the addition of the aforementioned two amino acids increased the acarbose production rate by 78 and 59%, respectively. Figure 7 Effect of amino acids on cell growth rate and acarbose production rate. To find candidate genes to be overexpressed to increase acarbose production, the strategy of FSEOF was applied. When the acarbose production rate was increased selleck chemicals gradually, four types of flux profiles were identified: increased, decreased, irregular, and unchanged flux. The flux profile is shown in Supplementary Data Sheet 4. The ratio of pentose phosphate pathway flux to glycolysis flux increased with increasing acarbose production. This simulation result reveals that acarbose production increased with the enhancement of the pentose phosphate pathway and the inhibition of glycolysis. Thus, genes in the pentose phosphate pathway, such as ACPL_1861, ACPL_6461, ACPL_1328, were candidates for overexpression to improve acarbose production. In addition, the flux of the reaction catalyzed by alanine aminotransferase (EC: 2.6.1.2), which forms glutamate, increased with increasing acarbose production. Thus, ACPL_6750 was another candidate gene to be overexpressed. Meanwhile, oxygen flux was reduced during the whole FSEOF strategy. Therefore, to gain insight into the effect of the oxygen uptake rate on acarbose production, the influence of the oxygen uptake rate on acarbose formation was studied by robustness analysis in acarbose synthesis medium. When the maltose and ammonium uptake rate were constrained to be 0.269 and 0.330 mmol/g DCW/h, respectively, and biomass was constrained to 0.025 h?1, it was found that the acarbose production rate increased sharply when the oxygen uptake rate was in the range of 0�C0.2 mmol/g DCW/h (Figure ?(Figure8).8). However, when the oxygen uptake rate exceeded 0.2 mmol/g DCW/h, the acarbose production rate began to decrease, and eventually reached zero as the oxygen uptake rate increased.