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 success in the clinic, 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. Towards filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KSCLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer. Using E. coli as a heterologous host, we obtained soluble proteins in titers representing significant improvements over previous KSCLF 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 KrKSCLF 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. Given the potential impact of obtaining soluble core type II PKS biosynthetic enzymes to enable in vitro characterization studies, these results lay important groundwork for optimizing the interaction between KrKSCLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

中文翻译：

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扩大瓶颈：大肠埃希氏菌最小化II型聚酮化合物合酶的异种表达，纯化和鉴定

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产生的分子在临床上取得了显著成功，但这些酶的生物合成能力受到了以下方面的阻碍：1）无法在体外重构最小限度的PKS酶的生物活性，以及​​2）对II型的有限探索来自不同门的PKS。为了满足这一未满足的需求，我们表达，纯化和鉴定了来自Ktedonobacter racemifer的酮合酶链长因子（KSCLF）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，代表了相对于以前的KSCLF异源表达工作的重大改进。这些酶的特征表明，KrACP具有自我丙二酰化活性。对holo-KrACP和KrKSCLF的沉降速度分析超速离心（SV-AUC）分析表明，这些酶在体外不相互作用，表明这些蛋白的酰化状态可能在促进生物合成相关相互作用中起重要作用。鉴于获得可溶性II型核心PKS生物合成酶的潜在影响以进行体外表征研究，这些结果为优化KrKSCLF和KrACP之间的相互作用以及探索其他非II型放线菌PKS的生物合成潜力奠定了重要基础。提示这些蛋白质的酰化状态可能在促进生物合成相关相互作用中起重要作用。鉴于获得可溶性II型核心PKS生物合成酶的潜在影响以进行体外表征研究，这些结果为优化KrKSCLF与KrACP之间的相互作用以及探索其他II型非放线菌PKS的生物合成潜力奠定了重要基础。提示这些蛋白质的酰化状态可能在促进生物合成相关相互作用中起重要作用。鉴于获得可溶性II型核心PKS生物合成酶的潜在影响以进行体外表征研究，这些结果为优化KrKSCLF和KrACP之间的相互作用以及探索其他非II型放线菌PKS的生物合成潜力奠定了重要基础。