Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways.

Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses.

—Jang et al.

Butanol is naturally produced by some anaerobic bacteria, but the efficiency of its production could not match that of ethanol due to the high toxicity of butanol to host organisms and the production of byproducts such as acetone and organic acids. Over the past decades, many research groups extensively studied clostridial butanol producing organisms to achieve higher yield and titer, but the limited genetic modification tools and complex metabolic pathways of Clostridia hampered the successful development of an engineered strain capable of producing butanol at a higher yield and titer.

The Korean research team analyzed metabolic pathways leading to butanol production and found that two different solvent-forming pathways can be potentially employed. In one pathway, butanol is directly produced from carbon source (“hot channel”); in the other, butanol is converted from the acids produced earlier in fermentation process (the “cold channel”).

Using in silico modeling and simulation tools, the team demonstrated that the hot channel allows a much better approach to produce butanol compared with the cold channel. To reinforce a metabolic flux toward the hot channel for butanol production, they systematically engineered the metabolic network of a C. acetobutylicum strain.

To maximize butanol production through the hot channel, they simultaneously disrupted the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while overexpressing the adhE1D485G gene which encodes a mutated aldehyde/alcohol dehydrogenase.

The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered strain.

By reinforcing the direct butanol-forming flux in C. acetobutylicum, they produced 18.9 g/liter of butanol, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type.

Using a fed-batch culture of this engineered strain with in situ recovery, they produced 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h.

In summary, system-level analysis of butanol-forming routes in C. acetobutylicum suggested new metabolic characteristics of butanol production in this bacterium. The direct butanol-forming hot channel and acid-reassimilating cold channel cooperate in the production of butanol, while the former plays a more important role in enhanced butanol production.

Butanol production by C. acetobutylicum could be enhanced with respect to all important bioprocess objectives, including product concentration, yield, selectivity, and productivity, by reinforcing the direct butanol-forming hot channel rather than the traditionally well-known acid reassimilation (cold channel) pathways. The new butanol-forming metabolic characteristics described here, together with the metabolic engineering strategies based on these findings, would be valuable for the development of a superior C. acetobutylicum strain capable of highly efficient butanol production. Further metabolic engineering will focus on achieving higher butanol tolerance, increased carbon flux toward butanol, and further reduction of by-product formation.

—Jang et al.

This research was supported by the Technology Development Program to Solve Climate Changes from the Ministry of Education, Science and Technology (MEST), Korea, the National Research Foundation of Korea, the Advanced Biomass Center through the Global Frontier Research Program of the MEST, and by the EEWS program of KAIST.

Resources

• Jang Y-S, et al. (2012) Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum. mBio 3(5):e00314-12. doi: 10.1128/mBio.00314-12