| dc.description.abstract | Isopropyl-β-D-1-thiogalactopyranoside (IPTG) plays significant role in
initiating expression of recombinant genes in Escherichia coli under the control of
lac-derived promoter. The addition of IPTG is frequently performed in exponential
growth stage by using high concentrations as an effort to fully induce lac-derived
promoter. Although generally results in increasing recombinant genes expression,
metabolic load that occurs in host cells after induction leads to reduce cell growth,
in turn, lowering product yield. The objective of this study was to investigate the
effect of IPTG addition in terms of induction time and IPTG concentration in
recombinant E. coli culture to improve ethanol production under aerobic conditions.
This study was consisted of three experimental stage: effect of pdc and adhB
introduction, role of fdh reaction and formate availability, and effect of IPTG
induction on ethanol production. E. coli strains used in this study were BW25113
and BW25113Δpta. Plasmid pTadhB-pdc harboring ethanol producing genes of pdc
and adhB from Zymomonas mobilis; and pHfdh harboring NADH regeneration
gene of fdh from Mycobacterium vaccae, were used to transform E. coli strains.
Improvement of ethanol production was first investigated by introducing pdc and
adhB genes into E. coli BW25113; and then was further improved by introducing
fdh and ethanologenic Z. mobilis genes into E. coli BW25113Δpta, following 4 g
L-1 formate supplementation into cultivation medium. The effect of induction time
in recombinant E. coli BW25113/pHfdh/pTadhB-pdc culture was investigated by
IPTG addition at 0, 6, 12 and 18 h of cultivation time; while impact of inducer
concentration was investigated by addition of IPTG in the range concentration of
50-2000 μM at an optimum induction time.
Analysis of mRNA and ethanol showed pdc and adhB were successfully
expressed in recombinant strain BW25113/pTadhB-pdc, and led to induced two
fold greater ethanol compared to the parent strain BW25113 (0.2 g L-1 ethanol). The
ethanol production improvement was further observed at 1.10 g L-1 in recombinant
culture of BW25113Δpta/pHfdh/pTadhB-pdc by using 4 g L-1 formate
supplementation. IPTG induction in BW25113Δpta/pHfdh/pTadhB-pdc culture
showed significant higher ethanol production than non-induced culture. Addition
of IPTG at concentration up to 50 μM strongly led to ethanol production
improvement. However, higher IPTG concentrations could not further improve the
production of ethanol. This study demonstrated that induction time at 0 h by using
low concentration of IPTG at 50 μM is effective to improve ethanol production in
recombinant E. coli culture under aerobic conditions. | id |
