Optimization of cell-free protein synthesis by proteomics and metabolic engineering of Escherichia coli A19

Abstract

Cell-free protein synthesis (CFPS) has emerged as a standard protein production system over the last two decades. Due to its open nature and various methods of directly influencing protein expression, it has replaced or complemented in vivo expression systems, especially for the expression of toxins, membrane proteins and other difficult-to-express proteins. Despite the widespread use of CFPS, the main component of the system, an extract derived from the centrifugation of a bacterial lysate (S30 extract), still has not been defined thoroughly. S30 extract preparation often causes changes in protein composition, altering the original proteome of exponentially growing Escherichia coli (E. coli). To optimize CFPS in a rational manner, S30 extracts from the E. coli K12-derivative A19 were analyzed using a GeLC-MS approach. The S30 core proteome, consisting of 821 proteins detected in several replicates, was functionally integrated and categorized using GO terms, revealing the presence of complete pathways that can be explored for energy regeneration or precursor generation. To evaluate the effects of alternative growth conditions, S30 extracts derived from cells grown at SOS response-inducing conditions were analyzed by quantitative GeLC-MS using isotope-coded protein labeling (ICPL). These modified S30-S extracts contained 3-10-fold increased folding factors and were shown to improve the solubility and folding of difficult proteins. Therefore, the manipulation of the S30 extract proteome by modifying the cultivation conditions is an effective approach for the expression of challenging proteins. A second approach to improve CFPS productivity was the engineering of specific metabolic pathways through genetic modifications. Using the previously generated proteome as a guideline, 13 genes coding for various enzymes affecting protein, amino acid and mRNA stability were either tagged or knocked out in E. coli strains A19 and D10. After verifying the modifications by PCR and sequencing, the viability and fitness of the strains were examined. Additionally, the transcriptional profile of a heavily modified strain was compared with the original A19 strain, revealing highly coregulated transcriptome in response to the genetic modification. The amino acid concentrations of 19 amino acids were traced during a CFPS reaction, demonstrating that amino acids can be stabilized by genetic modifications. The engineered strains showed an increase in yield for some target proteins, highlighting the relevance of metabolic engineering when optimizing CFPS. Finally, one of the metabolically engineered strains was used as an extract source and combined with purified chaperones (DsbC, Skp and FkpA) to produce different antibody fragments. DsbC was the most important chaperone for Fab folding, whereas Skp and FkpA were beneficial to produce scFab.