<jats:sec><jats:label /><jats:p>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 <jats:italic>Actinosynnema pretiosum</jats:italic> subsp. <jats:italic>pretiosum</jats:italic> ATCC 31280. For titer improvement, gene <jats:italic>ansa30</jats:italic>, encoding for a glycosyltransferase competing for the <jats:italic>N</jats:italic>‐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 <jats:italic>N</jats:italic>‐methylation is a rate‐limiting step. Alternatively, when abundant upstream intermediate 19‐chloroproansamitocin was fed into a PKS mutant, 3‐<jats:italic>O</jats:italic>‐acylation was further identified along with the <jats:italic>N</jats:italic>‐methylation as the rate‐limiting steps. Subsequent overexpression of <jats:italic>N</jats:italic>‐methyltransferase gene <jats:italic>asm10</jats:italic> 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‐<jats:italic>O</jats:italic>‐acylation bottleneck was relieved by feeding with L‐valine to NXJ‐22, resulting in a 126% increase of AP‐3. Eventually, a combined <jats:italic>asm10</jats:italic> overexpression and supplementation of L‐methionine and L‐valine resulted in a 5‐fold increase of AP‐3, from 42 ± 2 mg L<jats:sup>−1</jats:sup> to 246 ± 6 mg L<jats:sup>−1</jats:sup>, without any noticeable accumulation of PND‐3.</jats:sec>