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.

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.

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.

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 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.

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.

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.

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.

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.