Tirkkonen Heli, Brown Katelyn V, Niemczura Magdalena, Faudemer Zélie, Brown Courtney, Ponomareva Larissa V, Helmy Yosra A, Thorson Jon S, Nybo S Eric, Metsä-Ketelä Mikko, Shaaban Khaled A
Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland.
Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States.
ACS Omega. 2023 Jun 1;8(23):21237-21253. doi: 10.1021/acsomega.3c02460. eCollection 2023 Jun 13.
Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2',3',4'-tri-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8--d-glucosyl-tetracenomycin C, 8--d-olivosyl-tetracenomycin C, 8--d-mycarosyl-tetracenomycin C, and 8--d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-4'-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8--d-fucosyl-tetracenomycin C, 8--d-allosyl-tetracenomycin C, and 8--d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.
四环素霉素和埃洛霉素是由几种放线菌产生的聚酮类天然产物，具有抗菌和抗癌活性。它们通过结合在大核糖体亚基的多肽出口通道中抑制核糖体翻译。四环素霉素和埃洛霉素的典型特征是具有一个共同的氧化修饰线性十酮酸核心，但它们的区别在于O-甲基化程度以及埃洛霉素8位上连接的2',3',4'-三甲基-α-L-鼠李糖的存在。TDP-L-鼠李糖供体向8-去甲基四环素霉素C苷元受体的转移由多特异性糖基转移酶ElmGT催化。ElmGT对许多TDP-脱氧糖底物向8-去甲基四环素霉素C的转移表现出显著的灵活性，包括TDP-2,6-二脱氧糖、TDP-2,3,6-三脱氧糖以及d-和l-构型的甲基支链脱氧糖。此前，我们开发了一种改良宿主M1146::cos16F4iE，它是一个稳定的整合体，含有8-去甲基四环素霉素C生物合成和ElmGT表达所需的基因。在这项工作中，我们开发了用于链霉菌属中脱氧糖生物合成代谢工程的BioBricks基因盒。作为概念验证，我们使用BioBricks表达平台对d-构型的TDP-脱氧糖进行生物合成工程改造，包括已知化合物8--d-葡萄糖基-四环素霉素C、8--d-橄榄糖基-四环素霉素C、8--d-肉豆蔻糖基-四环素霉素C和8--d-洋地黄毒糖基-四环素霉素C。此外，我们还生成了四种新的四环素霉素，包括一种用酮糖修饰的8-4'-酮基-d-洋地黄毒糖基-四环素霉素C，以及三种用6-脱氧糖修饰的，包括8--d-岩藻糖基-四环素霉素C、8--d-阿洛糖基-四环素霉素C和8--d-奎诺糖基-四环素霉素C。我们的工作证明了BioBricks克隆的可行性，它能够回收中间构建体，用于快速组装各种碳水化合物途径以及多种天然产物的糖基多样化。
