Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

诸如II型聚酮化合物合酶（PKS）之类的酶组装物可产生多种生物活性次级代谢产物。虽然II型PKS产生的分子已获得了巨大的临床成功，但这些酶的生物合成能力受到了以下阻碍：1）无法在体外重建最小的PKS酶的生物活性，以及2）限制了对多种II型PKS的探索门。为了满足这一未满足的需求，我们表达，纯化和表征了来自总状竞争杆菌（Kr）的酮合成酶链长因子（KS-CLF）和酰基载体蛋白（ACP ）。使用大肠杆菌作为异源宿主，我们获得了滴度可溶的蛋白，表示相对于先前的KS-CLF异源表达工作有所改进。这些酶的表征表明，Kr ACP具有自我丙二酰化活性。沉降速度分析超速离心（SV-AUC）的分析全息-氪ACP和氪KS-CLF表明，这些酶不相互作用体外，这表明这些蛋白质的酰化状态可能在推动生物合成相关的相互作用中发挥重要作用。这些结果为优化Kr KS-CLF和Kr之间的相互作用奠定了重要的基础。ACP，并探索其他II类非放线菌PKS的生物合成潜力。