원문정보
초록
영어
The production of anti-HIV peptide, T-20, in recombinant Escherichia coli was enhanced by
analysis of metabolic load. Under metabolic load, stringent response occurs. Stringent
response is a complicated prokaryotic response system regulates gene expression in
transcription level to survive in nutrient depleted condition. On this response, cell stops
producing of foreign proteins and switches the gene expression to its survival. Guanosine
tetraphosphate (ppGpp) is the signal and effector molecule of this response. By monitoring
the intracellular ppGpp level, the occurrence of stringent response and metabolic load can be
determined. For understanding of physiological change of cell under metabolic load,
proteome analysis was done for E.coli BL21/pET23a-G3T20 after induction. Significant
spots were selected and malonyl CoA-acyl carrier protein transacylase(FabD) was finally
chosen. E.coli BL21/pET23a-G3T20/pACYC-fabD of which FabD is enhanced was
constructed and fed-batch fermentation was performed. Introducing fabD increased the T-
20/total protein% and T-20 production but decreased the cell growth. Expression of fabD also
maintained the intracellular ppGpp at half of the control. It was seems that enhancing FabD
caused the decrease of intracellular ppGpp, and low level of ppGpp could not trigger
stringent response under metabolic load. As a consequence, in E.coli BL21/pET23a-
G3T20/pACYC-fabD, 6.14% of T-20/total protein% and 392.49 of T-20 production (cell
growth*T-20%) was obtained and these values were about 3.5 times higher than E.coli
BL21/pET23a-G3T20 which produced 1.77% of T-20/total protein% and 143.72 of T-20
production (cell growth*T-20%)