Engineering Clostridium acetobutylicum for alcohol production

Area of Science:

• Microbial Biotechnology
• Synthetic Biology
• Biofuel Production

Background:

• Clostridium acetobutylicum is utilized for butanol production via ABE fermentation, but acetone is a non-biofuel byproduct.
• Acetone byproduct reduces the efficiency and economic viability of butanol as a biofuel.

Purpose of the Study:

• To engineer Clostridium acetobutylicum for enhanced alcohol (butanol and ethanol) titers.
• To eliminate acetone production and improve strain robustness for biofuel applications.

Main Methods:

• Inactivated the adc gene to abolish acetone production and introduced glutathione biosynthesis genes (gshAB) for enhanced robustness.
• Utilized overlapping PCR to create an expression cassette (EC) for key alcohol biosynthesis genes (hbd, thl, crt, bcd).
• Amplified the Sol operon to express adhE and ctfAB genes, further optimizing metabolic flux.

Main Results:

• Reduced acetone production from 2.64±0.22 g/L to 0.15±0.08 g/L in the engineered strain 824adc::gsh.
• Increased butanol production from 5.17±0.26 g/L to 8.27±0.27 g/L in the engineered strain.
• Achieved final butanol and total alcohol titers of 14.86±0.26 g/L and 18.11±0.66 g/L, respectively, in the 824adc::gsh Sol-EC strain.
• Obtained butanol and alcohol yields of 0.336 g/g and 0.409 g/g, respectively.

Conclusions:

• The combined strategy of eliminating acetone production and enhancing alcohol biosynthesis pathways significantly increases butanol and total alcohol production in Clostridium acetobutylicum.
• This engineered strain shows improved robustness and higher yields, making it a promising candidate for efficient biofuel production.
• The study provides a viable metabolic engineering approach for optimizing microbial biofuel synthesis.