Derivatization of polyketides during their biosynthesis

聚酮类化合物是一类结构多样的天然产物，在治疗和临床应用中具有多种功能，如用于抗生素治疗的红霉素或具有免疫抑制特性的雷帕霉素。模块化聚酮合成酶(PKS)属于自然界中最大、最复杂的酶工厂。由于蛋白质组织和产品结构的严格相关性，它们也是生物工程的一个令人兴奋的目标。作为共线性原理，聚酮的每个化学特征都直接与一个特定的模块和结构域联系在一起(S)。从这一点来看，通过定向修饰模块PKS，定制合成具有改变或改进功能的聚酮似乎是可能的。在一种概念验证方法中，我们最近能够利用这种杂交蛋白来合成氟化大内酯和大环内酯类化合物。成功的合成是基于Fas转移酶的混杂和对PKS模块Debs M6催化域的固有底物耐受性。我们现在已经收集了关于混合方法在“Debs-M6”系统之外的适用性的初步数据：我们发现，混合设计可以转移到其他PKS模块，并且另一个PKS模块能够合成含氟大内酯。这些初步结果为Fas/PKS杂化材料在聚酮生物合成中的广泛应用提供了前景。在本项目中，我们主要讨论了Fas/PKS杂化的两个必要的进一步发展：(1)PKS接受双取代底物，如氟甲基-丙二酰辅酶A。在这里，我们寻求设计的PKS模块也接受二甲基丙二酰辅酶A和螺环丙二酰辅酶A作为底物，并能够获得宝石-二甲基化或螺环聚酮。甲基是药物化学中的重要修饰，也被称为“魔力甲基”。我们感兴趣的是螺环基序作为宝石-二甲基团的替代物。(2)MAT结构域具有高度的底物耐受性，在简单的反应环境中提供了极好的合成机会。MAT的广泛应用现在依赖于提高底物的选择性，在这里，最重要的是抑制与天然底物丙二酸基和甲基丙二酸辅酶A的竞争负载。在工作包2中，特定于底物的垫变体的文库将被进化。在这个项目的框架内，我们将使用KS和MAT变体通过化学酶合成来生产12元和14元的修饰大内酯。该项目的前景是工程杂交物在微生物宿主生物中的应用。

Polyketides are a structurally diverse class of natural products that comprise various functions in therapeutic and clinical applications, such as erythromycin, which is applied in antibiotic therapy, or rapamycin, which has immunosuppressant properties. Modular polyketide synthases (PKSs) belong to nature’s largest and most complex enzymatic factories. They are also an exciting target for bioengineering due to the strict correlation of protein organization and product structure. Known as the principle of co-linearity, each chemical feature of a polyketide is directly linked to a specific module and domain(s). In this light, custom synthesis of polyketides with altered or improved functions seems possible through directed modifications of modular PKSs. In a proof-of-concept approach, we were recently able to harness such a hybrid protein for the chemoenzymatic synthesis of fluorinated macrolactones and macrolides. The successful synthesis was based on the promiscuity of the transferase from FAS and an intrinsic substrate tolerance of the catalytic domains of the PKS module DEBS M6. We have now collected preliminary data to the applicability of the hybrid approach beyond the "DEBS-M6" system: We found out that the hybrid design can be transferred to other PKS modules and that another PKS module is capable of synthesizing fluorinated macrolactones. These preliminary results offer the prospect of the broad application of FAS/PKS hybrids in polyketide biosynthesis. In the project presented, we primarily address two necessary further developments for the broad application of FAS/PKS hybrids: (1) PKSs accept disubstituted substrates, as exemplified by fluoromethyl-malonyl-CoA. Here, we seek to engineer PKS modules that also accept dimethyl-malonyl-CoA and spirocyclo-malonyl-CoA as substrates, and enable access to gem-dimethylated or spirocyclic polyketides. Methyl groups are important modifications in medicinal chemistry and are also known as "magic methyl". We are interested in the spirocyclic motifs as surrogates of gem-dimethyl groups. (2) The MAT domain is highly substrate tolerant and offers fantastic synthetic opportunities in simple reaction environments. The broad application of MAT now depends on improved substrate selectivity, and, here, above all on the suppression of competitive loading with the natural substrates, malonyl- and methylmalonyl-CoA. In work package 2, libraries of substrate-specific MAT variants are to be evolved. Within the framework of this project, we will produce 12- and 14-membered modified macrolactones via chemoenzymatic synthesis using the KS and MAT variants. The perspective of this project is the application of engineered hybrids in microbial host organisms.