A team at the Korea Advanced Institute of Science and Technology (KAIST) has developed a a novel strategy for microbial gasoline production through the metabolic engineering of Escherichia coli. The team engineered engineered platform E. coli strains that are capable of producing short-chain alkanes (SCAs; i.e., gasoline); free fatty acids (FFAs); fatty esters; and fatty alcohols via the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway.
As reported in their paper in Nature, the final engineered strain produced up to 580.8 mg per liter of SCAs consisting of nonane (327.8 mg l−1), dodecane (136.5 mg l−1), tridecane (64.8 mg l−1), 2-methyl-dodecane (42.8 mg l−1) and tetradecane (8.9 mg l−1), together with small amounts of other hydrocarbons.
Gasoline, the petroleum-derived product that is most widely used as a fuel for transportation, is a mixture of hydrocarbons, additives, and blending agents. Gasoline has a combination of straight-chain and branched-chain alkanes (hydrocarbons) consisted of 4-12 carbon atoms linked by direct carbon-carbon bonds.
Previously, through metabolic engineering of Escherichia coli (E. coli), there have been a few research results on the production of long-chain alkanes, which consist of 13-17 carbon atoms, suitable for replacing diesel. However, there has been no report on the microbial production of short-chain alkanes, a possible substitute for gasoline.
Bio-based sustainable production of fuels has been attracting increasing interest for our sustainable future. Hydrocarbon, such as alkane or alkene, is of particular interest owing to its potential to be used as an advanced biofuel that is similar to the petro-based fuels currently in use and superior to other biofuels in many aspects, including its high energy content (for example, it has a 30% higher energy content than ethanol). There have been a few reports on the bio-based production of C13–C17 long-chain hydrocarbons for substituting for diesel. Microbial production of up to 300 mg l-1 of long-chain hydrocarbons, mainly pentadecane and heptadecane, was achieved by using an engineered E. coli strain harbouring a cyanobacterial alkane biosynthesis operon encoding acyl-ACP reductase and aldehyde decarbonylase. Another study also reported production of even or odd numbered long-chain alkanes in E. coli by the overexpression of the Bacillus subtilis fabH gene. In these studies, hydrocarbons were produced by decarbonylation of fatty aldehydes, which are directly generated from fatty acyl-ACPs. More recently, long-chain alkanes were produced from fatty acids by using fatty acid reductase and aldehyde decarbonylase.
Petrol, a mixture of C4–C12 short-chain hydrocarbons (SCHCs), is a liquid fuel primarily used in internal combustion engines. Although short-chain alcohols were produced to substitute for petrol, they are inferior to petrol in their fuel properties. Thus, it is of great interest to produce SCHCs directly that have the potential to be used directly as petrol. However, there has been no report so far about the production of such SCHCs by microbial fermentation. This seems to be because most of the bacterial fatty acids identified are C14–C18 long-chain ones. Here we report the development of engineered E. coli strains capable of producing SCAs suitable for petrol by engineering fatty acid biosynthesis and degradation pathways. This was achieved, in a different way from previous studies on the production of long-chain hydrocarbons, by introducing a new pathway involving a mutant fatty acyl-ACP thioesterase, fatty acyl-CoA synthetase, fatty acyl-CoA reductase and fatty aldehyde decarbonylase into engineered E. coli supporting generation of short-chain fatty acyl-ACPs.—Choi and Lee (2013)
The research team engineered the fatty acid metabolism to provide the fatty acid derivatives that are shorter than normal intracellular fatty acid metabolites, and introduced a novel synthetic pathway for the biosynthesis of short-chain alkanes. This allowed the development of platform E. coli strain capable of producing gasoline for the first time. Furthermore, this platform strain, if desired, can be modified to produce other products such as short-chain fatty esters and short-chain fatty alcohols.
In this paper, the Korean researchers described detailed strategies for:
screening of enzymes associated with the production of fatty acids;
engineering of enzymes and fatty acid biosynthetic pathways to concentrate carbon flux towards the short-chain fatty acid production; and
converting short-chain fatty acids to their corresponding alkanes (gasoline) by introducing a novel synthetic pathway and optimization of culture conditions.
The research team also showed the possibility of producing fatty esters and alcohols by introducing responsible enzymes into the same platform strain.
It is only the beginning of the work towards sustainable production of gasoline. The titer is rather low due to the low metabolic flux towards the formation of short-chain fatty acids and their derivatives. We are currently working on increasing the titer, yield and productivity of bio-gasoline. Nonetheless, we are pleased to report, for the first time, the production of gasoline through the metabolic engineering of E. coli, which we hope will serve as a basis for the metabolic engineering of microorganisms to produce fuels and chemicals from renewable resources.—Professor Sang Yup Lee
This research was supported by the Advanced Biomass Research and Development Center of Korea (ABC-2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF), Republic of Korea. Systems metabolic engineering work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) by MSIP through NRF.
Yong Jun Choi & Sang Yup Lee (2013) “Microbial production of short-chain alkanes” Nature doi: 10.1038/nature12536