Identification and Engineering of Post-PKS Modification Bottlenecks for Ansamitocin P-3 Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280.

The type-I polyketide ansamitocin P-3 (AP-3) is a potent antitumor agent. Its production is most likely hampered by the required multiple substrate supplies and complicated post-PKS modifications in Actinosynnema pretiosum subsp. pretiosum ATCC 31280. For titer improvement, gene ansa30, encoding for a glycosyltransferase competing for the N-demethyl-AP-3 (PND-3) intermediate for AP-3 biosynthesis, was initially inactivated. In the mutant NXJ-22, the AP-3 titer was increased by 66% along with an obvious accumulation of PND-3, indicating that the N-methylation is a rate-limiting step. Alternatively, when abundant upstream intermediate 19-chloroproansamitocin was fed into a PKS mutant, 3-O-acylation was further identified along with the N-methylation as the rate-limiting steps. Subsequent overexpression of N-methyltransferase gene asm10 in NXJ-22 resulted in a 93% increase of AP-3 and a corresponding 92% decrease of PND-3. Additional supplementation of L-methionine, the precursor for SAM biosynthesis, substantially decreased the accumulation of PND-3. In parallel, the 3-O-acylation bottleneck was relieved by feeding with L-valine to NXJ-22, resulting in a 126% increase of AP-3. Eventually, a combined asm10 overexpression and supplementation of L-methionine and L-valine resulted in a 5-fold increase of AP-3, from 42 ± 2 mg L-1 to 246 ± 6 mg L-1 , without any noticeable accumulation of PND-3.