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. The second example of improved expression from yeast is the following: The yeast Pichia pastoris KM71H (MutS) is an efficient producer of hard-to-express proteins such as the membrane protein P-glycoprotein (Pgp), an ATP-powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and -ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and -ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400-fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris MutS strain and suggests an expression procedure for hard-to-express proteins from P. pastoris.