Ansamitocin P-3 (AP-3) shows strong anticancer effects and has used as a payload for antibody–drug conjugates. Our previous study have shown that although genetically engineered Actinosynnema pretiosum strains with enhanced UDP-glucose (UDPG) biosynthesis displayed improved AP-3 production compared to the wild-type strain, the increase in yield was far from meeting the industrial demand. In this study, comparative metabolomics analysis complemented with quantitative real-time PCR analysis was performed for the wild-type strain and two mutants (OpgmOugp, ΔzwfΔgnd) to identify possible metabolic bottlenecks and non-intuitive targets for further enhancement of AP-3 production. We observed that enhancing intracellular UDPG availability facilitated the accumulation of intracellular N-demethyl-AP-3 and AP-3, where the transporting of them outside the cell still needs to be developed. We also found that the UDPG biosynthesis was closely associated with the availability of fructose in the medium and a suitable fructose feeding strategy could promote the further improvement of AP-3 titer. In addition, pathway abundance analysis revealed that undesired fatty acid accumulation and down-regulation of amino acid metabolism may be unfavorable for ansamitocin biosynthesis in later stage of production. These results indicate that genetic modification of the UDPG biosynthetic pathways may have pleiotropic effects on AP-3 production. Efforts must be made to eliminate these newly identified metabolic bottlenecks to boost AP-3 production in A. pretiosum.

Ansamitocin P-3（AP-3）具有很强的抗癌作用，已用作抗体-药物偶联物的有效负载。我们以前的研究表明，尽管与野生型菌株相比，具有增强的UDP-葡萄糖（UDPG）生物合成功能的遗传工程化的猕猴桃菌株显示出改善的AP-3产量，但产量的增加远不能满足工业需求。在这项研究中，比较代谢分析补充有对野生型菌株和两种突变体（O进行定量实时PCR分析PGM ø UGP，Δ ZWF Δ GND），以确定可能的代谢瓶颈和非直观目标，以进一步提高AP-3的产量。我们观察到增强细胞内UDPG的利用率促进了细胞内N的积累-demethyl-AP-3和AP-3，仍然需要开发它们在细胞外的转运。我们还发现，UDPG的生物合成与培养基中果糖的可用性密切相关，合适的果糖喂养策略可以促进AP-3滴度的进一步提高。此外，途径丰度分析表明，不希望的脂肪酸积累和氨基酸代谢的下调可能不利于生产后期安沙霉素的生物合成。这些结果表明，UDPG生物合成途径的遗传修饰可能对AP-3产生多效性。必须努力消除这些新近发现的代谢瓶颈，以提高A. pretiosum中AP-3的产量。