Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli.

BACKGROUND: A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. RESULTS: We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. CONCLUSION: The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

A novel knock out strategy to enhance recombinant protein expression in Escherichia coli.

BACKGROUND: The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression. RESULTS: We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Yp/x) was observed in a BW25113ΔcysJ knock out (Yp/x 0.57) and BW25113ΔelaA (Yp/x 0.49), whereas the Yp/x of the control W3110 strain was 0.08 and BW25113 was 0.16. Double knock out combinations were then created from the ten best performing single knock outs leading to a further enhancement in expression levels. Out of 45 double knock outs created, BW25113ΔelaAΔyhbC (Yp/x 0.7) and BW25113ΔcysJΔyhbC (Yp/x 0.64) showed the highest increase in product yield compared to the single gene mutant strains. We confirmed the improved performance of these knock outs by testing and obtaining higher levels of recombinant asparaginase expression, a system better suited for analysing sustained expression since it gets exported to the extracellular medium. CONCLUSION: Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.

Metabolic engineering of Escherichia coli W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol.

L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in Escherichia coli is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.

Synthesis of dopamine in E. coli using plasmid-based expression system and its marked effect on host growth profiles.

L-Dopa and dopamine are important pathway intermediates toward the synthesis of catecholamine such as epinephrine and norepinephrine from amino acid L-tyrosine. Dopamine, secreted from dopaminergic nerve cells, serves as an important neurotransmitter. We report the synthesis of dopamine by extending the aromatic amino acid pathway of Escherichia coli DH5α by the expression of 4-hydroxyphenylacetate-3-hydrolase (HpaBC) from E. coli and an engineered dopa decarboxylase (DDC) from pig kidney cell. The activity of HpaBC and DDC require 200 µM iron supplementation and 50 µM vitamin B6, respectively as additives to the growth media. The maximum concentration of L-dopa and dopamine obtained from the broth was around 26 and 27 mg/L after 24 hr of separate shake flask studies. We observed that in the presence of dopamine synthesized in vivo host growth was remarkably enhanced. These observations lead us to an interesting finding about the role of these catecholamines on bacterial growth. It is clear that synthesis of dopamine in vivo actually promotes growth much efficiently as compared to when dopamine is added to the system from outside. From HPLC and GC-MS data it was further observed that L-dopa was stable within the observable time of experiments whereas dopamine actually was subjected to degradation via oxidation and host consumption.

Genome engineering for improved recombinant protein expression in Escherichia coli.

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.

Efficient 3-Hydroxybutyrate Production by Quiescent Escherichia coli Microbial Cell Factories is Facilitated by Indole-Induced Proteomic and Metabolomic Changes.

The authors show that quiescent (Q-Cell) Escherichia coli cultures can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater specific productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Q-cells can be created by using the proton ionophore indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks because less metabolic effort is diverted to surplus biomass production. However, the reason for the increased productivity of cells in the quiescent state was previously unknown. In this study, proteome expression patterns between wild-type and Q-cell cultures show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Metabolomic data reveal the accumulation of acetyl-coenzyme A and phosphoenolpyruvate: excellent starting points for high-value chemical production. We demonstrate the exploitation of these accumulated metabolites by engineering a simple pathway for 3HB production from acetyl-coenzyme A. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g L-1 of 3HB compared to 18.6 g L-1 in the control. Q-cells therefore have great potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.

Indole generates quiescent and metabolically active Escherichia coli cultures.

An inherent problem with bacterial cell factories used to produce recombinant proteins or metabolites is that resources are channeled into unwanted biomass as well as product. Over several years, attempts have been made to increase efficiency by unlinking biomass and product generation. One example was the quiescent cell (Q-Cell) expression system that generated non-growing but metabolically active Escherichia coli by over-expressing a regulatory RNA (Rcd) in a defined genetic background. Although effective at increasing the efficiency with which resources are converted to product, the technical complexity of the Rcd-based Q-Cell system limited its use. We describe here an alternative method for generating Q-Cells by the direct addition of indole, or related indole derivatives, to the culture medium of an E. coli strain carrying defined mutations in the hns gene. This simple and effective approach is shown to be functional in both shake-flask and fermenter culture. The cells remain metabolically active and analysis of their performance in the fermenter suggests that they may be particularly suitable for the production of cellular metabolites.

Supplementation of substrate uptake gene enhances the expression of rhIFN-ß in high cell density fed-batch cultures of Escherichia coli.

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the "ara" promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-ß (rhIFN-ß) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-ß in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK (+)) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-ß in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-ß was obtained with the modified strain which was 35 % higher than the control.

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data.

Fed batch cultures expressing recombinant interferon beta under the T7 promoter were run with different exponential feeding rates of a complex substrate and induced at varying cell densities. Post-induction profiles of the specific product formation rates showed a strong dependence on the specific growth rate with the maximum product yield obtained at 0.2 h(-1). A study of the relative transcriptomic profiles as a function of pre-induction µ was therefore done to provide insight into the role of cellular physiology in enhancing recombinant protein expression. Hierarchical clustering analysis of the significantly regulated genes allowed us to identify biologically important groups of genes which fall under specific master regulators. The groups were: rpoH, ArcB, CreB, Lrp, RelA, Fis and Hfq. The response of these regulators, which exert a feedback control on the growth and product formation rates correlated well with the expression levels obtained. Thus at the optimum pre-induction µ, the alternative sigma factors and ribosomal machinery genes did not get depressed till the 6th hour post-induction unlike at other specific growth rates, demonstrating a critical role for the genes in sustaining recombinant protein expression.

Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli.

There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-ß), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-ß and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.