DOI: https://doi.org/10.61435/ijred.2026.62506
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Optimization of fermentation conditions using T7 promoter-engineered Escherichia coli for isobutanol production

1Doctoral Program of Biology, School of Life Science and Technology, Bandung Institute of Technology, Indonesia

2School of Life Science and Technology, Bandung Institute of Technology, Indonesia

3Program of Biotechnology, Faculty of Science and Technology, Muhammadiyah University of Bandung, Indonesia

4 Center of Excellence for Nutraceuticals, Bioscience and Biotechnology Research Center. Bandung Institute of Technology, Indonesia

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Received: 14 Mar 2026; Revised: 19 Aug 2026; Accepted: 15 Oct 2026; Available online: 23 Oct 2026; Published: 1 Nov 2026.
Editor(s): H Hadiyanto
Open Access Copyright (c) 2026 The Author(s). Published by Centre of Biomass and Renewable Energy (CBIORE)
Creative Commons License This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

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Abstract

Isobutanol is a promising biofuel alternative to ethanol due to its higher energy density, lower hygroscopicity, and greater compatibility with existing fuel infrastructure. However, microbial isobutanol production remains constrained by inefficient heterologous protein expression and suboptimal fermentation conditions. This study aimed to improve isobutanol production by combining promoter engineering with fermentation parameter optimization in recombinant Escherichia coli BL21(DE3). The lac promoter in the isobutanol biosynthetic plasmid, containing alsS, ilvC, ilvD, and kivd, was replaced with the T7 promoter. Protein expression was evaluated using SDS–PAGE, while isobutanol production was quantified by GC–FID. Subsequently, pH, temperature, and fermentation time were optimized using a three-factor Box–Behnken response surface methodology design comprising 15 experimental runs. The T7 promoter enhanced recombinant protein expression and increased isobutanol production compared with the lac promoter system, with concentrations of 0.2766 and 0.1784 g/L, respectively. The response surface model was statistically significant (F = 7.48, p = 0.0053) and explained 67.12% of the variability in isobutanol production (R² = 0.6712). Temperature was the only statistically significant individual factor, with lower temperatures generally favoring isobutanol formation. The highest experimentally observed isobutanol titer was 0.313 g/L, corresponding to a glucose-to-isobutanol yield of 0.0281 g/g. Validation under the optimized conditions was performed through time-course fermentation measurements to monitor biomass growth and isobutanol accumulation. The results demonstrate that combining T7-based expression with fermentation optimization can improve isobutanol production in recombinant E. coli, although the relatively low predicted R² indicates that additional biological and process-related factors remain to be addressed.

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Keywords: Isobutanol; Box-Behnken; Biofuel; Escherichia coli; T7 Promoter

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Section: Original Research Article
Language : EN
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