期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 114, 期 12, 页码 2794-2806出版社
WILEY
DOI: 10.1002/bit.26396
关键词
Actinosynnema pretiosum; ansamitocin P-3; fermentation technology; metabolic engineering; secondary metabolite; traditional mutation
资金
- National Natural Science Foundation of China [31370083]
- Science and Technology Commission of Shanghai Municipality [14XD1402600]
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.
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