Understanding metabolism and stress conditions of recombinant microorganisms

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

A general approach for improved expression from E. coli is described below: The concern is that the recombinant proteins expression triggers a stress response which downregulates key metabolic pathway genes cause a decline in cellular health and feedback inhibition growth and protein expression. Instead of upregulating these downregulated genes or improve transcription rates by better vector design, an innovative strategy would be to block this stress response and ensuring a sustained level of protein expression. Results: We postulated that the genes which are commonly up-regulated following induction may play a role of signaling messengers in mounting cellular stress response. We identified those genes which have no known downstream regulatees and created knockouts strains which were then tested for GFP expression. Many of these knockouts showed significantly higher expression levels which also sustained for longer periods. The highest product yield (Yp/x) was observed in a BW25113cysJ knockout (Yp/x 0.57) and BW25113elaA(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, the strains BW25113elaAyhbC (Yp/x 0.7) and BW25113cysJyhbC (Yp/x 0.64) showed the highest increase in product yield compared with their single gene mutant strains. The improved performance of these knockouts was confirmed by testing and obtaining higher levels of recombinant asparaginase expression. Additional project was associated with iron and oxygen effect on e coli metabolism and gene expression. Aerobic Escherichia coli growth at restricted iron concentrations ( 1.75 0.04 M) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems