Combination of traditional mutation and metabolic engineering to enhance ansamitocin P‐3 production in Actinosynnema pretiosum

Abstract Ansamitocin P‐3 (AP‐3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP‐3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP‐3 biosynthesis in Actinosynnema pretiosum . First, a mutant strain M was isolated by N ‐methyl‐ N' ‐nitro‐ N ‐nitrosoguanidine mutation, which could produce AP‐3 almost threefold that of wild type (WT) in 48 deep‐well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13‐17 in the M strain, a further 60% increase of AP‐3 production in 250‐ml shake flasks was achieved in the engineered strain M‐ asmUdpg:asm13‐17 compared to the M strain, and its maximum AP‐3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP‐3 biosynthesis pathway were significantly increased in the M and M‐ asmUdpg:asm13‐17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab‐scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP‐3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.