Metabolic Engineering of Gas-Fermenting Clostridium ljungdahlii for Efficient Co-production of Isopropanol, 3-Hydroxybutyrate, and Ethanol.

Rational design and modification of autotrophic bacteria to efficiently produce high-value chemicals and biofuels are crucial for establishing a sustainable and economically viable process for one-carbon (C1) source utilization, which, however, remains a challenge in metabolic engineering. In this study, autotrophic Clostridium ljungdahlii was metabolically engineered to efficiently co-produce three important bulk chemicals, isopropanol, 3-hydroxybutyrate (3-HB), and ethanol (together, IHE), using syngas (CO2/CO). An artificial isopropanol-producing pathway was first constructed and optimized in C. ljungdahlii to achieve an efficient production of isopropanol and an unexpected product, 3-HB. Based on this finding, an endogenous active dehydrogenase capable of converting acetoacetate to 3-HB was identified in C. ljungdahlii, thereby revealing an efficient 3-HB-producing pathway. The engineered strain was further optimized to reassimilate acetic acid and synthesize 3-HB by introducing heterologous functional genes. Finally, the best-performing strain was able to produce 13.4, 3.0, and 28.4 g/L of isopropanol, 3-HB, and ethanol, respectively, in continuous gas fermentation. Therefore, this work represents remarkable progress in microbial production of bulk chemicals using C1 gases.

Enhanced alcohol titre and ratio in carbon monoxide-rich off-gas fermentation of Clostridium carboxidivorans through combination of trace metals optimization with variable-temperature cultivation.

Bioconversion of C1 gases to produce chemicals has good application prospects. Here, the combination of trace metals optimization using a statistical method with variable-temperature cultivation was used to enhance alcohol synthesis during CO-rich off-gas fermentation by Clostridium carboxidivorans P7. Based on ATCC medium 1754, the optimum concentration of the trace metals was found to be 5-fold Ni2+, Co2+, SeO42+, and WO42+; 3.48-fold Cu2+; 0.55-fold MoO42+; 0.5-fold Zn2+ and (NH4)2SO4·FeSO4·6H2O; and additional 44.32µM FeCl3·6H2O. The production of alcohol and organic acid changed to 4.40g/L and 0.50g/L from 2.16g/L and 2.37g/L, respectively, yielding an increase of alcohol-to-product ratio from 47.7% to 89.8%. By fermenting with the optimized medium and timed control of the incubation temperature (37°C [0-24h]-25°C [24-144h]), the alcohol titre further increased to 6.97g/L with 1.67g/L butanol and 1.33g/L hexanol, exceeding those previously reported for strain P7.