Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli
Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli
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DOI：
10.1016/j.bmc.2020.115686
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发表时间：
2020-10-15
影响因子：
3.5
通讯作者：
Charkoudian, Louise K.
中科院分区：
文献类型：
--
作者：
Klein, Joshua G.;Wu, Yang;Charkoudian, Louise K.
酶组件，如II型聚酮合成酶(PKS)，可产生广泛的生物活性次生代谢物。虽然由II型PKS产生的分子已经在临床上取得了显著的成功，但这些酶的生物合成能力受到以下因素的阻碍：1)无法在体外重建最小的PKS酶的生物活性；2)从不同的门中提取II型PKS的探索有限。为了开始满足这一未得到满足的需求，我们表达、纯化和鉴定了总状克氏杆菌(Kr)的酮合酶链长因子(KS-CLF)和酰基载体蛋白(ACP)。使用大肠杆菌作为异源宿主，我们获得了滴度较以前的KS-CLF异源表达努力有所改善的可溶性蛋白。对这些酶的表征表明，KrACP具有自丙二酰化活性。HALO-KrACP和KrKS-CLF的沉降速度分析超速离心法(SV-AUC)分析表明，这些酶在体外不相互作用，这表明这些蛋白质的酰化状态可能在促进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KrKS-CLF和KrACP之间的相互作用以及探索其他非放线菌II型PKS的生物合成潜力奠定了重要的基础。
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.