Identifying genomic targets for protein over-expression by "omics" analysis of Quiescent Escherichia coli cultures.

BACKGROUND: A cellular stress response is triggered upon induction of recombinant protein expression which feedback inhibits both growth as well as protein synthesis. In order to separate these two effects, it was decided to study "quiescent cultures" which continue to be metabolically active and express recombinant proteins even after growth cessation. The idea was to identify and up-regulate genes which are responsible for protein synthesis in the absence of growth. This would ensure that, even if growth were adversely affected post induction, there would be no attendant reduction in the protein expression capability of the cells. This strategy allowed us to design host strains, which did not grow better post induction but had significantly higher levels of protein expression. RESULTS: A quiescent Escherichia coli culture, which is able to sustain recombinant protein expression in the absence of growth, was analyzed by transcriptomic and proteomic profiling. Many genes involved in carbon utilization, biosynthesis of building blocks and stress protection were found to be up-regulated in the quiescent phase. Analysis of the global regulators showed that fis, which tends to get down-regulated as the cells enter stationary phase, remained up-regulated throughout the non-growing quiescent phase. The downstream genes regulated by fis like carB, fadB, nrfA, narH and queA were also up-regulated in the quiescent phase which could be the reason behind the higher metabolic activity and protein expression ability of these non-growing cells. To test this hypothesis, we co-expressed fis in a control culture expressing recombinant L-asparaginase and observed a significantly higher buildup of L-asparaginase in the culture medium. CONCLUSIONS: This work represents an important breakthrough in the design of a superior host platform where a gene not directly associated with protein synthesis was used to generate a phenotype having higher protein expression capability. Many alternative gene targets were also identified which may have beneficial effects on expression ability.

An inverse metabolic engineering approach for the design of an improved host platform for over-expression of recombinant proteins in Escherichia coli.

BACKGROUND: A useful goal for metabolic engineering would be to generate non-growing but metabolically active quiescent cells which would divert the metabolic fluxes towards product formation rather than growth. However, for products like recombinant proteins, which are intricately coupled to the growth process it is difficult to identify the genes that need to be knocked-out/knocked-in to get this desired phenotype. To circumvent this we adopted an inverse metabolic engineering strategy which would screen for the desired phenotype and thus help in the identification of genetic targets which need to be modified to get overproducers of recombinant protein. Such quiescent cells would obviate the need for high cell density cultures and increase the operational life span of bioprocesses. RESULTS: A novel strategy for generating a library, consisting of randomly down regulated metabolic pathways in E. coli was designed by cloning small genomic DNA fragments in expression vectors. Some of these DNA fragments got inserted in the reverse orientation thereby generating anti-sense RNA upon induction. These anti-sense fragments would hybridize to the sense mRNA of specific genes leading to gene 'silencing'. This library was first screened for slow growth phenotype and subsequently for enhanced over-expression ability. Using Green Fluorescent Protein (GFP) as a reporter protein on second plasmid, we were able to identify metabolic blocks which led to significant increase in expression levels. Thus down-regulating the ribB gene (3, 4 dihydroxy-2-butanone-4-phosphate synthase) led to a 7 fold increase in specific product yields while down regulating the gene kdpD (histidine kinase) led to 3.2 fold increase in specific yields. CONCLUSION: We have designed a high throughput screening approach which is a useful tool in the repertoire of reverse metabolic engineering strategies for the generation of improved hosts for recombinant protein expression.

Co-expressing Leucine Responsive Regulatory protein (Lrp) enhances recombinant L-Asparaginase-II production in Escherichia coli.

Over expression of recombinant proteins triggers a cellular stress response (CSR) that down-regulates numerous genes that have a key role in sustaining expression. Instead of trying to individually up-regulate these genes we hypothesized that a superior strategy would be to modulate the expression of global regulators that control the expression of many such downstream genes. Transcriptomic profiling of post induction cultures expressing recombinant asparaginase in Escherichia coli showed the down-regulation of several critical genes many of which were under the control of the global regulator lrp which is known to have a significant impact on both amino acid metabolism and protein translation. Therefore, to ameliorate the deleterious effects of the CSR we decided to supplement the activity of lrp using plasmid-based co-expression. We observed that the test culture containing an additional plasmid expressing lrp under the arabinose promoter gave a 50% higher yield of recombinant L-Asparaginase after 32 h in batch culture compared to the control, which had only one plasmid expressing the recombinant protein. This approach helped us design a better performing strain, which could sustain expression rates for a significantly longer time period. This work illustrates that modifying the expression of regulatory genes could serve as a better strategy to prevent the reprogramming of the cellular machinery which is the hallmark of the CSR and help in the design better hosts for recombinant protein expression.

Genomics and transcriptomics analysis reveals the mechanism of isobutanol tolerance of a laboratory evolved Lactococcus lactis strain.

Isobutanol, in spite of its significant superiority over ethanol as a biofuel, remains commercially non-viable due to the non-availability of a suitable chassis which can handle the solvent toxicity associated with its production. To meet this challenge, we chose Lactococcus lactis which is known for its ability to handle environmental stress and carried out Adaptive laboratory evolution (ALE) in a continuous stirred tank reactor (CSTR) to evolve an isobutanol tolerant strain. The strain was grown for more than 60 days (> 250 generations) while gradually increasing the selection pressure, i.e. isobutanol concentration, in the feed. This led to the evolution of a strain that had an exceptionally high tolerance of up to 40 g/l of isobutanol even though a scanning electron microscope (SEM) study as well as analysis of membrane potential revealed only minor changes in cellular morphology. Whole genome sequencing which was done to confirm the strain integrity also showed comparatively few mutations in the evolved strain. However, the criticality of these mutations was reflected in major changes that occurred in the transcriptome, where gene expression levels from a wide range of categories that involved membrane transport, amino acid metabolism, sugar uptake and cell wall synthesis were significantly altered. Analysing the synergistic effect of these changes that lead to the complex phenotype of isobutanol tolerance can help in the construction of better host platforms for isobutanol production.

Designing an Escherichia coli Strain for Phenylalanine Overproduction by Metabolic Engineering.

The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG fbr performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications. Knocking in the tktA gene under a tyrR promoter and knocking out pykF further increased phenylalanine production demonstrating that the supply of precursor via PEP and E4P is also a rate-limiting step. Finally, we tested the role of global regulators on this deregulated pathway and found that Fis overexpression helps in both enhancing and sustaining the flux through this pathway. This work opens up the possibility of using global regulators in synergy with pathway-specific modifications to enhance product yields.

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 microg/mL in the control to 40 microg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 microg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.

Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system.

The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.5 mg g(-1) DCW h(-1) at a dilution rate (D) of 0.3 h(-1). The maximum volumetric product concentration achieved at this dilution rate was 474 mg l(-1), which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h(-1) and 0.4 h(-1) respectively. The specific product yield (Y(P/x)) peaked at 138 mg g(-1) DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q(p) was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l(-1) with a specific hGM-CSF yield (Y(P/x)) of 107 mg g(-1) DCW.

Production and purification of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) from high cell density cultures of Pichia pastoris.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which has been used as a therapeutic agent in clinical cases like neutropenia. In this study, we report the production of recombinant human GM-CSF in the methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. Recombinant P. pastoris GS115 cells were grown in fed batch cultures to obtain a biomass density of 55.6 gDCW L(-1) and a high volumetric activity of 131 mg L(-1) of GM-CSF. The protein migrated as a diffuse band on SDS-PAGE at the range of 28-35 kDa indicating differential glycosylation. The secreted protein was purified to 95% in two steps using cation exchange and size exclusion chromatography.