Rational approach to improve ansamitocin P‐3 production by integrating pathway engineering and substrate feeding in Actinosynnema pretiosum

: Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is an important antitumor agent for cancer treatment, but its market supply suffers from a low production titer. The role of AP-3 unusual glycolate unit supply on its biosynthesis was investigated in this work by overexpressing the responsible gene cluster asm13-17 in A. pretiosum (WT). As a result, the accumulation of AP-3 and its intermediate glyceryl-S-ACP in the asm13-17-overexpressed strain (Oasm13-17) versus WT was enhanced by 1.94 and 1.49-fold, respectively. To provide a higher supply of another precursor 3-amino-5-hydroxybenzoic acid, asmUdpg was also overexpressed in Oasm13-17 (Oasm13-17:asmUdpg), and an improved AP-3 titer of 680.5 mg/L was achieved in shake flasks. To further enhance the AP-3 titer, a rational fed-batch strategy was developed in bioreactor fermentation of Oasm13-17:asmUdpg; and by pulse feeding 15 g/L fructose and 1.64 g/L isobutanol at 60, 96, and 120 hr, the AP-3 production level reached 757.7 mg/L, which is much higher than ever reported in bioreactors. This work demonstrated that a rational approach combining precursor pathway engineering with substrate feeding was very effective in enhancing the AP-3 titer, and this enabling methodology would be helpful to industrial production of this eye-catching drug.