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.