Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity.
Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity.
复制标题
DOI：
10.1186/s12934-021-01578-3
复制
发表时间：
2021-04-21
影响因子：
6.4
通讯作者：
Ding X
中科院分区：
文献类型：
--
作者：
Wang H;Liang J;Yue Q;Li L;Shi Y;Chen G;Li YZ;Bian X;Zhang Y;Zhao G;Ding X
聚酮合酶（PKS）包括酮合酶（KS）、酰基转移酶（AT）和酰基载体蛋白（ACP）结构域，以催化聚酮链的延长。一些 PKS 还含有酮还原酶 (KR)、脱水酶 (DH) 和烯酰还原酶 (ER) 结构域作为修饰结构域。催化结构域的插入、缺失或取代可能导致新型聚酮化合物衍生物的产生或所需产物的积累。埃坡霉素是 16 元大环内酯类化合物，已被用作抗癌药物。埃坡霉素 PKS (EPOAT4) 模块 4 AT 结构域的底物混杂导致埃坡霉素混合物的产生；该结构域的取代可能会改变埃博霉素的比例。此外，埃坡霉素 PKS 的模块 9 中有两个休眠结构域。消除这些冗余域以生成更简单、更高效的装配线是一个理想的目标。模块 4 的替换极大地降低了埃坡霉素 PKS 的活性。然而，通过仔细设计KS-AT接头和后AT接头，用EPOAT2、EPOAT6、EPOAT7或EPOAT8（特别是掺入甲基丙二酸单酰辅酶A（MMCoA））取代EPOAT4，显着增加了埃博霉素D（4）与埃博霉素C（3）的比例（最高比例为4:3 = 4.6:1），而亲本菌株 Schlegelella brevitalea 104-1 中的 4:3 为 1.4:1。我们还通过将 EPOAT4 与 EPOAT3、EPOAT5 或 EPOAT9 交换来获得三个菌株，它们特异性地掺入了丙二酰辅酶 A (MCoA)。这些菌株仅产生埃坡霉素C，且产量比亲本菌株104-1增加了1.8倍。此外，AT 结构域中 5 个残基的突变将 Ser310 确定为 EPOAT4 中 MMCoA 识别的关键因素。然后，His308 突变为缬氨酸或酪氨酸，结合 Phe310 突变为丝氨酸，进一步改变了产物比率。同时，我们成功删除了非活性模块 9 DH 和 ER 结构域，并通过约 25 个残基连接子将 ΨKR 结构域与 KR 结构域融合，生成高效且简化的埃坡霉素 PKS。这些结果表明，催化结构域的取代和删除有效地产生了所需的化合物，并且结构域之间的接头的选择对于维持完整的 PKS 催化活性至关重要。在线版本包含可在 10.1186/s12934-021-01578-3 获取的补充材料。
Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity. The online version contains supplementary material available at 10.1186/s12934-021-01578-3.
登录
查看更多内容
影响因子：
4.8
作者：
Klaus, Maja;Ostrowski, Matthew P.;Khosla, Chaitan
通讯作者：
Khosla, Chaitan
影响因子：
14.8
作者：
Kelley LA;Mezulis S;Yates CM;Wass MN;Sternberg MJ
通讯作者：
Sternberg MJ
影响因子：
56.9
作者：
Maier, Timm;Leibundgut, Marc;Ban, Nenad
通讯作者：
Ban, Nenad
影响因子：
11.5
作者：
Lechleider, Robert J.;Kaminskas, Edvardas;Pazdur, Richard
通讯作者：
Pazdur, Richard
影响因子：
8
作者：
Rittner, Alexander;Paithankar, Karthik S.;Grininger, Martin
通讯作者：
Grininger, Martin