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A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production
bioRxiv - Synthetic Biology Pub Date : 2020-04-05 , DOI: 10.1101/2020.04.05.026393 David C. Garcia , Jaime Lorenzo N. Dinglasan , Him Shrestha , Paul E. Abraham , Robert L. Hettich , Mitchel J. Doktycz
bioRxiv - Synthetic Biology Pub Date : 2020-04-05 , DOI: 10.1101/2020.04.05.026393 David C. Garcia , Jaime Lorenzo N. Dinglasan , Him Shrestha , Paul E. Abraham , Robert L. Hettich , Mitchel J. Doktycz
Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell’s capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction.
中文翻译:
用于提高无细胞代谢产物生产的裂解物蛋白质组工程策略
无细胞系统提供了利用多种生物的新陈代谢潜力的重要机会。去除细胞环境可提供无需维持细胞活力即可生产生物产品的能力,并使代谢工程师能够探索新型化学转化系统。粗提物可维持细胞的大部分功能。但是,只有有限的工具可用于工程化用于无细胞系统的提取物的内容。因此,我们充分利用粗提物进行无细胞代谢工程的潜力的能力受到限制。在这里,我们采用多重自动化基因组工程(MAGE)标记蛋白质,以从粗提物中选择性去除蛋白质,从而专门指导化学生产。在不显着影响细胞生长的情况下,可能对中央代谢进行特殊编辑。丙酮酸降解酶的选择性去除被证明导致与非工程提取物相比,工程化的粗产物裂解物能够使丙酮酸产量增加10到20倍。所描述的方法将系统和合成生物学的工具融合在一起,以将无细胞代谢工程发展为生物原型和生物生产的实用平台。
更新日期:2020-04-05
中文翻译:
用于提高无细胞代谢产物生产的裂解物蛋白质组工程策略
无细胞系统提供了利用多种生物的新陈代谢潜力的重要机会。去除细胞环境可提供无需维持细胞活力即可生产生物产品的能力,并使代谢工程师能够探索新型化学转化系统。粗提物可维持细胞的大部分功能。但是,只有有限的工具可用于工程化用于无细胞系统的提取物的内容。因此,我们充分利用粗提物进行无细胞代谢工程的潜力的能力受到限制。在这里,我们采用多重自动化基因组工程(MAGE)标记蛋白质,以从粗提物中选择性去除蛋白质,从而专门指导化学生产。在不显着影响细胞生长的情况下,可能对中央代谢进行特殊编辑。丙酮酸降解酶的选择性去除被证明导致与非工程提取物相比,工程化的粗产物裂解物能够使丙酮酸产量增加10到20倍。所描述的方法将系统和合成生物学的工具融合在一起,以将无细胞代谢工程发展为生物原型和生物生产的实用平台。