Abstract
A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.
Highlights
Host cell engineering has emerged as a powerful tool for designing microbial platforms targeted at improved metabolite production
The use of metabolic engineering strategies to remove the bottlenecks in recombinant protein production identified by analyzing this stress response has helped in improving the supply of precursors like NADPH, modification of global stress regulators and increasing the flux of the down regulated metabolic pathways including that of substrate uptake [135,136,137,138,139,140]
Since some fragments get inserted in the opposite orientation, they act as an anti-sense RNA and create a library of down-regulated pathways which can be screened for improved recombinant protein expression [178]
Summary
Host cell engineering has emerged as a powerful tool for designing microbial platforms targeted at improved metabolite production. Most importantly recombinant protein expression triggers a cellular stress response which feedback inhibits both growth and product formation, by lowering substrate uptake rates, down-regulating the ribosomal machinery and biosynthesis of ATP (Figure 2). The use of metabolic engineering strategies to remove the bottlenecks in recombinant protein production identified by analyzing this stress response has helped in improving the supply of precursors like NADPH, modification of global stress regulators and increasing the flux of the down regulated metabolic pathways including that of substrate uptake [135,136,137,138,139,140]. Since some fragments get inserted in the opposite orientation, they act as an anti-sense RNA and create a library of down-regulated pathways which can be screened for improved recombinant protein expression [178] This has been extended to the use of synthetic Small regulatory RNAs (sRNAs) which helps in the modulation of gene expression [169]. Apart from growth profiling, these systems can do online monitoring of fluorescence, pH, dissolved oxygen and NADH [185] and are reviewed in [191,192,193,194,195]
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