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

Abstract 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 (O asm13‐17 ) 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 O asm13‐17 (O asm13‐17: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 O asm13‐17: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.