Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression

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.