Improvement Of Ethanol Production By Inducer Addition In Recombinant Escherichia Coli Culture Under Aerobic Conditions

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.