A BioBricks toolbox for metabolic engineering of the tetracenomycin pathway.
A BioBricks toolbox for metabolic engineering of the tetracenomycin pathway.
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DOI：
10.1002/biot.202100371
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发表时间：
2022-03
影响因子：
4.7
通讯作者：
Nybo SE
中科院分区：
文献类型：
--
作者：
Nguyen JT;Riebschleger KK;Brown KV;Gorgijevska NM;Nybo SE
四环霉素是芳香族抗癌聚酮化合物，通过与核糖体大亚基结合来抑制肽翻译。在此，我们在异源宿主天蓝色链霉菌M1146中表达了elloramycin生物合成基因簇，以促进下游生产四环素类似物。我们开发了一个BioBricks®遗传工具箱，用于S. coelicolor M1146：：cos16F4iE.我们克隆了一系列基于VWB、TG 1和SV 1整合酶系统的整合载体，以询问染色体中的基因表达。我们通过基因工程改造了三种不同的基因构建体来调节四环霉素的生物合成：（1）来自专性需氧玻璃球菌的vhb血红蛋白，以提高氧的利用率;（2）accA 2BE乙酰辅酶A羧化酶，以增强丙二酰辅酶A的缩合;（3）最后，sco 6196酰基转移酶，其是负责将三酰基甘油动员到乙酰辅酶A的β-氧化机制的“代谢调节开关”。此外，我们还从拟无枝酸菌A23中改造了tcmO 8-O-甲基转移酶和新鉴定的tcmD 12-O-甲基转移酶，以产生四烯霉素C和X。我们还将tcmO甲基转移酶与加氧酶urdE共表达，以产生类似物6-羟基-四环霉素C。总之，该系统与BioBricks® [RFC 10]克隆标准兼容，用于天蓝色链霉菌M1146：：cos 16 F4 iE代谢工程的多个基因集的共表达。该生产平台改善了对有效类似物（如四环霉素X）的获取，并为通过组合生物合成生产新的四环霉素奠定了基础。我们开发了整合载体、启动子和生物合成基因的BioBricks®工具箱，用于工程改造四环霉素途径。我们通过过表达编码玻璃球菌血红蛋白（vhb）、天蓝色链霉菌乙酰辅酶A羧化酶（accA 2BE）和天蓝色链霉菌脂肪酰辅酶A合酶（sco 6196）的基因来提高四环霉素聚酮的生产，以产生能够在摇瓶发酵中生产400 mg/L 8-去甲基-四环霉素C的菌株。最后，我们通过过表达最近表征的tcmD 12-O-甲基转移酶，将8-去甲基-四环霉素C途径转向生产更有效的类似物，四环霉素X，从而为通过组合生物合成产生新的四环霉素奠定了基础。
The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. We developed a BioBricks® genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: 1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a “metabolic regulatory switch” responsible for mobilizing triacylglycerols to β-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Altogether, this system is compatible with the BioBricks® [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE. This production platform improves access to potent analogs, such as tetracenomycin X, and sets the stage for the production of new tetracenomycins via combinatorial biosynthesis. We developed a BioBricks® toolbox of integrating vectors, promoters, and biosynthetic genes for engineering the tetracenomycin pathway. We improved tetracenomycin polyketide production by overexpressing genes encoding Vitreoscilla stercoraria hemoglobin (vhb), Streptomyces coelicolor acetyl-CoA carboxylase (accA2BE), and a Streptomyces coelicolor fatty acyl-CoA synthase (sco6196) to generate a strain capable of producing 400 mg/L 8-demethyl-tetracenomycin C in shake flask fermentations. Lastly, we diverted the 8-demethyl-tetracenomycin C pathway towards production of the more potent analog, tetracenomycin X, by overexpressing the recently characterized tcmD 12-O-methyltransferase, thereby setting the stage for generation of new tetracenomycins via combinatorial biosynthesis.
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影响因子：
2.7
作者：
Nybo, S. Eric;Shabaan, Khaled A.;Kharel, Madan K.;Sutardjo, Happy;Salas, Jose A.;Mendez, Carmen;Rohr, Juergen
通讯作者：
Rohr, Juergen
影响因子：
2.1
作者：
Morita, Kentaro;Yamamoto, Tomoyuki;Takahashi, Hideo
通讯作者：
Takahashi, Hideo
影响因子：
5.1
作者：
Fischer, C;Rodríguez, L;Rohr, J
通讯作者：
Rohr, J
DOI：
10.1073/pnas.0709223105
发表时间：
2008-04-08
影响因子：
11.1
作者：
Ames, Brian Douglas;Korman, Tyler Paz;Tsai, Shiou-Chuan
通讯作者：
Tsai, Shiou-Chuan
影响因子：
3.3
作者：
DRAUTZ, H;REUSCHENBACH, P;ZEECK, A
通讯作者：
ZEECK, A