A team of UCLA researchers led by Professor James Liao have engineered Escherichia coli bacteria to produce 1-butanol with high titer (30 g/L) and high yield (70 - 88% of theoretical production) anaerobically, comparable to or exceeding the levels demonstrated by native producers. An open access paper on their work appears in the journal Applied Environmental Microbiology.
For the study, Liao and his team initially constructed a 1-butanol biochemical pathway in E. coli, a microbe that doesn’t naturally produce 1-butanol, but found that production levels were limited. However, after adding metabolic driving forces to the pathway by genetically engineering the metabolism, the researchers witnessed a tenfold increase in the production of 1-butanol. The metabolic driving forces pushed the carbon flux to 1-butanol.
1-Butanol, a potential fuel substitute and an important C4 chemical feedstock, is naturally synthesized by Clostridium species using a pathway that involves multiple coenzyme A (CoA) activated intermediates (hereafter called the CoA-dependent pathway). Various attempts have been reported to transfer the CoA-dependent 1-butanol pathway to more tractable organisms, such as Escherichia coli (0.55-1.2 g/L), Saccharomyces cerevisiae (2.5 mg/L), Lactobacillus brevis (300 mg/L), Pseudomonas putida (580 mg/L) and Bacillus subtilis (120 mg/L). These low titers of heterologous 1-butanol production demonstrate the difficulty in transferring this pathway to non-native hosts and are in sharp contrast to the high titers of related compounds produced by recombinant E. coli such as ethanol (50 g/L), isobutanol (20 - 50 g/L), and isopropanol (40 - 140 g/L) using related pathways.
Examination of these high titer productions revealed that all of the pathways involve a decarboxylation reaction near the end products, in which the irreversible release of CO2 serves as a driving force to pull fluxes to the desired compounds. Such a strategy is also present in fatty acid synthesis, which involves decarboxylation of malonyl-CoA in the chain elongation step. In contrast, when the Clostridial CoA-dependent 1-butanol pathway is transferred to E. coli, no significant driving force exists to direct the carbon flux through the five reversible steps starting from acetyl-CoA.
We reason that a significant driving force is necessary to achieve high titer production whereas driving forces are not as important in proof-of-concept productions...metabolic engineering or synthetic biology strategies for high titer production can be summarized and recast in two steps: 1) creating a driving force, and 2) coupling the driving force to the desired pathway.—Shen et al.
While certain microbes, including species of the bacteria Clostridium, naturally produce 1-butanol, Liao’s team used E. coli because it is easier to manipulate and has been used industrially in producing various chemicals.
By using E. coli, we can make it produce only the compound with no other byproducts. With native producing organisms like Clostridium, which naturally produces 1-butanol, there are other byproducts that would add cost to the separation process.—James Liao
The next step in the research, the researchers say, will be to transfer the study to industry for the development of a more robust industrial process. The study was funded by the KAITEKI Institute Inc. of Japan, a strategic arm of Mitsubishi Chemical Holdings Corp., Japan’s largest chemical company.
Shen, Claire R., Lan, Ethan I., Dekishima, Yasumasa, Baez, Antonino, Cho, Kwang Myung, Liao, James C. (2011) High titer anaerobic 1-butanol synthesis in Escherichia coli enabled by driving forces Appl. Environ. Microbiol. doi: 10.1128/AEM.03034-10