Isobutanol Production by Autotrophic Acetogenic Bacteria

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Biotechnology

Background:

• Acetogenic bacteria like Acetobacterium woodii and Clostridium ljungdahlii are crucial for converting syngas into valuable chemicals.
• Isobutanol is a promising biofuel and chemical feedstock, but efficient microbial production remains a challenge.
• Understanding and engineering specific metabolic pathways are key to enhancing microbial production of target compounds.

Purpose of the Study:

• To clone and express two distinct isobutanol synthesis pathways in A. woodii and C. ljungdahlii.
• To evaluate the efficiency of different gene clusters and enzyme combinations for isobutanol production.
• To optimize production through substrate supplementation and gene knockout strategies.

Main Methods:

• Cloning and expression of isobutanol synthesis pathways (using genes like kor and adhE2, or ketoisovalerate decarboxylase and alcohol dehydrogenase) into A. woodii and C. ljungdahlii.
• Testing different gene clusters from Clostridium thermocellum (kor2, kor3).
• Supplementation with ketoisovalerate and inactivation of the ilvE gene in C. ljungdahlii to enhance carbon flux and production.

Main Results:

• Traces of isobutanol were detected in recombinant A. woodii strains using the ketoisovalerate ferredoxin oxidoreductase (Kor) pathway.
• Supplementation with ketoisovalerate significantly increased isobutanol production in both A. woodii (up to 2.9 mM) and C. ljungdahlii (up to 1.5 mM).
• Inactivation of the ilvE gene in C. ljungdahlii further improved isobutanol yields, reaching 2.4 mM under specific conditions.

Conclusions:

• The Kor pathway, particularly kor3, showed promise for isobutanol production in A. woodii.
• Ketoisovalerate supplementation is a critical factor for enhancing isobutanol yields in both engineered bacterial hosts.
• Metabolic engineering strategies, including gene knockout, can further optimize microbial production of isobutanol.