Cross-tolerance and antagonistic pleiotropy have been observed between different complex phenotypes in microbial systems. These relationships between adaptive landscapes are important for the design of industrially relevant strains, which are generally subjected to multiple stressors. In our previous work, we evolved Escherichia coli for enhanced tolerance to the biofuel n-butanol and discovered a molecular mechanism of n-butanol tolerance that also conferred tolerance to the cationic antimicrobial peptide polymyxin B in one specific lineage (green fluorescent protein [GFP] labeled) in the evolved population. In this work, we aim to identify additional mechanisms of n-butanol tolerance in an independent lineage (yellow fluorescent protein [YFP] labeled) from the same evolved population and to further explore potential cross-tolerance and antagonistic pleiotropy between n-butanol tolerance and other industrially relevant stressors. Analysis of the transcriptome data of the YFP-labeled mutants allowed us to discover additional membrane-related and osmotic stress-related genes that confer n-butanol tolerance in E. coli. Interestingly, the n-butanol resistance mechanisms conferred by the membrane-related genes appear to be specific to n-butanol and are in many cases antagonistic with isobutanol and ethanol. Furthermore, the YFP-labeled mutants showed cross-tolerance between n-butanol and osmotic stress, while the GFP-labeled mutants showed antagonistic pleiotropy between n-butanol and osmotic stress tolerance.

Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1–xylose medium.

Strain tolerance to toxic metabolites remains an important issue in the production of biofuels. Here we examined the impact of overexpressing the heterologous groESL chaperone from Clostridium acetobutylicum to enhance the tolerance of Escherichia coli against several stressors. Strain tolerance was identified using strain maximum specific growth rate (μ) and strain growth after a period of solvent exposure. In comparison with control strain, the groESL overexpressing strain yielded a 27 % increase in growth under 0.8 % (v/v) butanol, a 9 % increase under 1 % (v/v) butanol, and a 64 % increase under 1.75 (g/l) acetate. Moreover, after 10 h, groESL overexpression resulted in increase in relative tolerance of 58 % compared with control strain under 0.8 % (v/v) butanol, 56 % increase under 1 % (v/v) butanol, 42 % increase under 1 % (v/v) isobutanol, 36 % increase under 4 % (v/v) ethanol, 58 % increase under 1.75 (g/l) acetate. These data demonstrate that overexpression of the groESL from C. acetobutylicum in E. coli increased tolerance to several stressors. Solvent tolerant strain of E. coli was developed to be used as a basic strain for biofuel production.

Isoprene represents a key building block for the production of valuable materials such as latex, synthetic rubber or pharmaceutical precursors and serves as basis for advanced biofuel production. To enhance the production of the volatile natural hydrocarbon isoprene, released by plants, animals and bacteria, the Kudzu isoprene synthase (kIspS) gene has been heterologously expressed in Bacillus subtilis DSM 402 and Bacillus licheniformis DSM 13 using the pHT01 vector. As control, the heterologous expression of KIspS in E. coli BL21 (DE3) with the pET28b vector was used. Isoprene production was analyzed using Gas Chromatography Flame Ionization Detector. The highest isoprene production was observed by recombinant B. subtilis harboring the pHT01-kIspS plasmid which produced 1434.3 μg/L (1275 µg/L/OD) isoprene. This is threefold higher than the wild type which produced 388 μg/L (370 μg/L/OD) isoprene, when both incubated at 30 °C for 48 h and induced with 0.1 mM IPTG. Additionally, recombinant B. subtilis produced fivefold higher than the recombinant B. licheniformis, which produced 437.2 μg/L (249 μg/L/OD) isoprene when incubated at 37 °C for 48 h induced with 0.1 mM IPTG. This is the first report of optimized isoprene production in B. licheniformis. However, recombinant B. licheniformis showed less isoprene production. Therefore, recombinant B. subtilis is considered as a versatile host for heterologous production of isoprene.