细菌天然产物（NPs）是药物和生物杀虫剂的不可或缺的来源。异源表达是发现细菌NPs和高效生物合成有价值NPs的基本方法，但革兰氏阴性细菌NPs的底盘仍然不足。在本研究中，我们通过引入整合位点（attB）来构建Burkholderiales突变株 Burkholderia gladioli Δgbn::attB，以使其失活本地 gladiolin（gbn）生物合成基因簇，稳定大型外源基因簇并减少本地代谢产物谱。通过对兼性近缘 Burkholderiales 衍生的抗肿瘤聚酮（PKs）rhizoxins、兼性远缘分离的甲氧西林耐药金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus）抗生素WAP-8294As和兼性 δ- 蛋白菌纪的抗肿瘤 PKs disorazols 进行异源表达，成功地培养并成功产生了高价值的NPs，表明该菌株具有成为革兰氏阴性细菌NPs的潜在底盘的能力。我们通过启动子插入和前体途径过表达进一步提高了 WAP-8294As 的产量，基于对此菌株的异源表达。该研究为基因组挖掘、高效生产和分子工程的细菌NPs提供了强大的菌株底盘。

Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and the efficient biosynthesis of valuable NPs, but the chassis for Gram-negative bacterial NPs remains inadequate. In this study, we built a Burkholderiales mutantBurkholderia gladioli Δgbn::attB by introducing an integrated site (attB) to inactivate the native gladiolin (gbn) biosynthetic gene cluster, which stabilizes large foreign gene clusters and reduces the native metabolite profile. The growth and successful heterologous production of high-value NPs such as phylogenetically close Burkholderiales-derived antitumor polyketides (PKs) rhizoxins, phylogenetically distant Gammaproteobacteria-derived anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotics WAP-8294As, and Deltaproteobacteria-derived antitumor PKs disorazols demonstrate that this strain is a potential chassis for Gram-negative bacterial NPs. We further improved the yields of WAP-8294As through promoter insertions and precursor pathway overexpression based on heterologous expression in this strain. This study provides a robust bacterial chassis for genome mining, efficient production, and molecular engineering of bacterial NPs.