Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression
Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression
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DOI：
10.1021/acssynbio.0c00283
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发表时间：
2020-10-16
影响因子：
4.7
通讯作者：
Oza, Javin P.
中科院分区：
文献类型：
--
作者：
Levine, Max Z.;So, Byungcheol;Oza, Javin P.
无细胞蛋白质合成（CFPS）平台已经经历了许多工作流程的改进，以实现研究，生物制造和教育中的各种应用。基于大肠杆菌细胞提取物的平台因其经济性和多功能性而被广泛采用。细胞的上游加工以产生粗细胞裂解物仍然是时间密集型的，并且在技术上是细微差别的，代表了与生物技术相关的最大成本来源之一。为了克服这些局限性，我们通过开发用于CFPS的长效自诱导培养基配方来改进工艺，该配方避免了接种和收获之间的人为干预。无细胞自诱导（CFAI）培养基支持从接近生长稳定期的高细胞密度培养物中生产稳健的细胞提取物。结果，细胞的总质量和所得的提取物体积增加了400%，同时保持报告蛋白sfGFP（> lmg/mL）的稳健反应产率。值得注意的是，CFAI工作流程允许用户在24小时内从划线板上的细胞完成CFPS反应。CFAI工作流程独特地使我们能够阐明与在传统2X YTPG培养基中生长至稳定期的细胞相关的CFPS中的代谢限制。代谢组学分析表明，基于CFAI的提取物由于改善了能量代谢和氧化还原平衡而克服了这些限制。这里报道的进展揭示了与高度活跃的CFPS反应相关的代谢，并为未来调整CFPS系统代谢的努力提供了信息。此外，我们预计CFPS在时间和成本效益方面的改进将增加简单性和可重复性，减少对实施CFPS感兴趣的新研究人员的障碍。
Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, and education. The Escherichia coli cell extract-based platform has been broadly adopted due to its affordability and versatility. The upstream processing of cells to generate crude cell lysate remains time-intensive and technically nuanced, representing one of the largest sources of cost associated with the biotechnology. To overcome these limitations, we have improved the processes by developing a long-lasting autoinduction media formulation for CFPS that obviates human intervention between inoculation and harvest. The cell-free autoinduction (CFAI) media supports the production of robust cell extracts from high cell density cultures nearing the stationary phase of growth. As a result, the total mass of cells and the resulting extract volume obtained increases by 400% while maintaining robust reaction yields of reporter protein, sfGFP (>1 mg/mL). Notably, the CFAI workflow allows users to go from cells on a streak plate to completing CFPS reactions within 24 h. The CFAI workflow uniquely enabled us to elucidate the metabolic limits in CFPS associated with cells grown to stationary phase in the traditional 2X YTPG media. Metabolomics analysis demonstrates that CFAI-based extracts overcome these limits due to improved energy metabolism and redox balance. The advances reported here shed new light on the metabolism associated with highly active CFPS reactions and inform future efforts to tune the metabolism in CFPS systems. Additionally, we anticipate that the improvements in the time and cost-efficiency of CFPS will increase the simplicity and reproducibility, reducing the barriers for new researchers interested in implementing CFPS.