A pressure test to make 10 molecules in 90 days: External evaluation of methods to engineer biology,Journal of the American Chemical Society

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A pressure test to make 10 molecules in 90 days: External evaluation of methods to engineer biology
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2018-02-26 , DOI: 10.1021/jacs.7b13292
Arturo Casini _{1,

2} , Fang-Yuan Chang _{1,

3} , Raissa Eluere _{1,

2} , Andrew M King _{1,

3} , Eric M Young _{1,

3} , Quentin M Dudley _{1,

4} , Ashty Karim _{1,

4} , Katelin Pratt _{1,

2} , Cassandra Bristol _{1,

2} , Anthony Forget _{1,

2,

3} , Amar Ghodasara ₃ , Robert Warden-Rothman _{1,

3} , Rui Gan _{1,

4} , Alexander Cristofaro _{1,

3} , Amin Espah Borujeni _{1,

3} , Min-Hyung Ryu ₃ , Jian Li ₄ , Yong-Chan Kwon ₄ , He Wang ₄ , Evangelos Tatsis ₅ , Carlos Rodriguez-Lopez ₅ , Sarah O'Connor ₅ , Marnix H Medema ₆ , Michael A Fischbach _{1,

7} , Michael C Jewett _{1,

4} , Christopher Voigt _{1,

2,

3} , D Benjamin Gordon _{1,

2,

3}

Affiliation

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

中文翻译：

在 90 天内制造 10 个分子的压力测试：工程生物学方法的外部评估

基因工程或“生物铸造厂”的集中设施提供了设计生物体的潜力，以满足医学、农业、工业和国防领域的新兴需求。该领域的技术进步很快，但很难衡量当前的能力或找出项目之间的差距。为此，我们通过定时“压力测试”对我们的铸造厂进行了评估，其中给予了 3 个月的时间来构建生物体，以提前生产出我们不知道的 10 个分子。通过应用多种新方法，我们在性能期间为 10 个目标中的 6 个生产了所需的分子或密切相关的分子，并在生产其他目标方面也取得了进展。具体来说，我们提高了 1-hexadecanol、pyrrolnitrin 和 pacidamycin D 的滴度，发现了强效抗菌剂背后的烯二炔弹头的新途径，建立了用于单萜生产的无细胞系统，产生了长春新碱生物合成的中间体，并在 DNA 库中编码了 7802 条可单独检索到 540 种双吲哚的途径。设计和构建了四氢呋喃和巴巴胺的途径，但毒性或分析工具阻碍了进一步的进展。总之，我们构建了 1.2 Mb DNA，构建了 215 个菌株，涵盖 5 个物种（酿酒酵母、大肠杆菌、白黄链霉菌、天蓝色链霉菌和白葡萄链霉菌），建立了两个无细胞系统，并进行了 690 种内部开发的检测方法。分子。并编码了 7802 条可单独检索的途径，通向 DNA 库中的 540 个双吲哚。设计和构建了四氢呋喃和巴巴胺的途径，但毒性或分析工具阻碍了进一步的进展。总之，我们构建了 1.2 Mb DNA，构建了 215 个菌株，涵盖 5 个物种（酿酒酵母、大肠杆菌、白黄链霉菌、天蓝色链霉菌和白葡萄链霉菌），建立了两个无细胞系统，并进行了 690 种内部开发的检测方法。分子。并编码了 7802 条可单独检索的途径，通向 DNA 库中的 540 个双吲哚。设计和构建了四氢呋喃和巴巴胺的途径，但毒性或分析工具阻碍了进一步的进展。总之，我们构建了 1.2 Mb DNA，构建了 215 个菌株，涵盖 5 个物种（酿酒酵母、大肠杆菌、白黄链霉菌、天蓝色链霉菌和白葡萄链霉菌），建立了两个无细胞系统，并进行了 690 种内部开发的检测方法。分子。

更新日期：2018-02-26

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