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

5 years ago

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

Jian-Jiang Zhong, Zhi-Gang Qian, Zhi-Qiang Du, Han Xiao, Yuan Zhang

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. Ansamitocin P-3 (AP-3) is approved as an important clinical drug of antibody conjugates for targeted cancer therapy, but there is a compelling need to enhance the antibiotic production for wide commercial application. Zhong's group at SJTU combined traditional mutation and metabolic engineering of Actinosynnema pretiosum, and significantly increased AP-3 fermentation titer, reaching nearly 600 mg/L, the highest as ever reported. Furthermore, insightful information on AP-3 biosynthesis was obtained and discussed by investigating both the gene transcription and intracellular intermediate levels.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/bit.26396