Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin.
Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin.
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非核糖体大环内酯肽缬氨霉素的体外全生物合成
DOI：
10.1016/j.ymben.2020.03.009
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发表时间：
2020-07
影响因子：
8.4
通讯作者：
Li J
中科院分区：
文献类型：
--
作者：
Zhuang L;Huang S;Liu WQ;Karim AS;Jewett MC;Li J
天然产品因其重要的药物特性（如抗病毒、抗菌和抗癌活性）而变得重要。 DNA测序的最新突破揭示了微生物基因组中编码了大量神秘的天然产物生物合成基因簇，例如链霉菌属物种的基因组。然而，从这些簇中获取化合物仍然具有挑战性，因为许多来源生物体是不可培养的，或者基因在实验室培养过程中是沉默的。为了应对这一挑战，我们开发了一个高效的无细胞平台，用于快速体外全生物合成非核糖体肽缬氨霉素作为模型。我们通过两种方式实现这一目标。首先，我们使用无细胞蛋白质合成（CFPS）系统在单锅反应中表达整个缬氨霉素生物合成基因簇（>19 kb），优化后产生约37 μg/L的缬氨霉素。其次，我们通过混合两种酶富集的细胞裂解物进行两阶段生物合成，将 CFPS 与无细胞代谢工程系统结合起来。该策略将缬氨霉素产量提高了约 5,000 倍，达到近 30 mg/L。我们期望无细胞生物合成系统将为体外表达、发现和表征感兴趣的天然产物基因簇提供新途径。
Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 μg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5,000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.
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影响因子：
4.6
作者：
Kwon YC;Jewett MC
通讯作者：
Jewett MC
影响因子：
4.7
作者：
Jaitzig, Jennifer;Li, Jian;Neubauer, Peter
通讯作者：
Neubauer, Peter
影响因子：
8.4
作者：
Karim, Ashty S.;Jewett, Michael C.
通讯作者：
Jewett, Michael C.
影响因子：
3.2
作者：
Dudley, Quentin M.;Nash, Connor J.;Jewett, Michael C.
通讯作者：
Jewett, Michael C.
无细胞的合成生物学，用于药物天然产物的体外生物合成。
影响因子：
4.8
作者：
Li J;Zhang L;Liu W
通讯作者：
Liu W