Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression.,ACS Synthetic Biology

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Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-08-24 , DOI: 10.1021/acssynbio.0c00283
Max Z Levine _{1,

2} , Byungcheol So _{2,

3} , Alissa C Mullin _{2,

3} , Rob Fanter ₄ , Kayla Dillard ₅ , Katharine R Watts _{2,

3} , Michael R La Frano _{5,

6} , Javin P Oza _{2,

3}

Affiliation

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 2× 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.

中文翻译：

通过生长培养基重新配制激活能量代谢使无细胞表达的 24 小时工作流程成为可能。

无细胞蛋白质合成 (CFPS) 平台已经经历了多次工作流程改进，以实现在研究、生物制造和教育中的多种应用。该大肠杆菌基于细胞提取物的平台因其经济性和多功能性而被广泛采用。细胞的上游处理以产生粗细胞裂解物仍然是时间密集型和技术上的细微差别，是与生物技术相关的最大成本来源之一。为了克服这些限制，我们通过为 CFPS 开发一种持久的自诱导培养基配方来改进流程，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。因此，获得的细胞总质量和所得提取物体积增加了 400%，同时保持了报告蛋白 sfGFP (>1 mg/mL) 的稳健反应产量。尤其，CFAI 工作流程允许用户在 24 小时内从条纹板上的细胞到完成 CFPS 反应。CFAI 工作流程独特地使我们能够阐明与在传统 2× 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，由于改善了能量代谢和氧化还原平衡，基于 CFAI 的提取物克服了这些限制。这里报告的进展为与高活性 CFPS 反应相关的代谢提供了新的线索，并为未来调整 CFPS 系统中的代谢的努力提供了信息。此外，我们预计 CFPS 在时间和成本效率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。

更新日期：2020-10-17

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