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KAIST announced a novel technology to produce gasoline by a metabolically engineered microorganism
A major scientific breakthrough in the development of renewable energy sources and other important chemicals; The research team succeeded in producing 580 mg of gasoline per liter of cultured broth by converting in vivo generated fatty acids
For many decades, we have been relying on fossil resources to produce liquid fuels such as gasoline, diesel, and many industrial and consumer chemicals for daily use. However, increasing strains on natural resources as well as environmental issues including global warming have triggered a strong interest in developing sustainable ways to obtain fuels and chemicals.
Gasoline, the petroleum-derived product that is most widely used as a fuel for transportation, is a mixture of hydrocarbons, additives, and blending agents. The hydrocarbons, called alkanes, consist only of carbon and hydrogen atoms. 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.
In the paper (entitled "Microbial Production of Short-chain Alkanes") published online in Nature on September 29, a Korean research team led by Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering at the Korea Advanced Institute of Science and Technology (KAIST) reported, for the first time, the development of a novel strategy for microbial gasoline production through metabolic engineering of E. coli.
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 1) screening of enzymes associated with the production of fatty acids, 2) engineering of enzymes and fatty acid biosynthetic pathways to concentrate carbon flux towards the short-chain fatty acid production, and 3) converting short-chain fatty acids to their corresponding alkanes (gasoline) by introducing a novel synthetic pathway and optimization of culture conditions. Furthermore, the research team showed the possibility of producing fatty esters and alcohols by introducing responsible enzymes into the same platform strain.
Professor Sang Yup Lee said, "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."
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.
Short-Chain Alkanes Generated from Renewable Biomass
This diagram shows the metabolic engineering of Escherichia coli for the production of short-chain alkanes (gasoline) from renewable biomass.
Nature Cover Page (September 29th, 2013)
2013.11.04 View 10911 -
A powerful strategy for developing microbial cell factories by employing synthetic small RNAs
The current systems for the production of chemicals, fuels and materials heavily rely on the use of fossil resources. Due to the increasing concerns on climate change and other environmental problems, however, there has been much interest in developing biorefineries for the production of such chemicals, fuels and materials from renewable resources. For the biorefineries to be competitive with the traditional fossil resource-based refineries, development of high performance microorganisms is the most important as it will affect the overall economics of the process most significantly. Metabolic engineering, which can be defined as purposeful modification of cellular metabolic and regulatory networks with an aim to improve the production of a desired product, has been successfully employed to improve the performance of the cell. However, it is not trivial to engineer the cellular metabolism and regulatory circuits in the cell due to their high complexity.
In metabolic engineering, it is important to find the genes that need to be amplified and attenuated in order to increase the product formation rate while minimizing the production of undesirable byproducts. Gene knock-out experiments are often performed to delete those metabolic fluxes that will consequently result in the increase of the desired product formation. However, gene knock-out experiments require much effort and time to perform, and are difficult to do for a large number of genes. Furthermore, the gene knock-out experiments performed in one strain cannot be transferred to another organism and thus the whole experimental process has to be repeated. This is a big problem in developing a high performance microbial cell factory because it is required to find the best platform strain among many different strains. Therefore, researchers have been eager to develop a strategy that allows rapid identification of multiple genes to be attenuated in multiple strains at the same time.
A Korean research team led by Distinguished Professor Sang Yup Lee at the Department of Chemical and Biomolecular Engineering from the Korea Advanced Institute of Science and Technology (KAIST) reported the development of a strategy for efficiently developing microbial cell factories by employing synthetic small RNAs (sRNAs). They first reported the development of such system in Nature Biotechnology last February. This strategy of employing synthetic sRNAs in metabolic engineering has been receiving great interest worldwide as it allows easy, rapid, high-throughput, tunable, and un-doable knock-down of multiple genes in multiple strains at the same time. The research team published a paper online on August 8 as a cover page (September issue) in Nature Protocols, describing the detailed strategy and protocol to employ synthetic sRNAs for metabolic engineering.
In this paper, researchers described the detailed step-by-step protocol for synthetic sRNA-based gene expression control, including the sRNA design principles. Tailor-made synthetic sRNAs can be easily manipulated by using conventional gene cloning method. The use of synthetic sRNAs for gene expression regulation provides several advantages such as portability, conditionality, and tunability in high-throughput experiments. Plasmid-based synthetic sRNA expression system does not leave any scar on the chromosome, and can be easily transferred to many other host strains to be examined. Thus, the construction of libraries and examination of different host strains are much easier than the conventional hard-coded gene manipulation systems. Also, the expression of genes can be conditionally repressed by controlling the production of synthetic sRNAs. Synthetic sRNAs possessing different repression efficiencies make it possible to finely tune the gene expression levels as well. Furthermore, synthetic sRNAs allow knock-down of the expression of essential genes, which was not possible by conventional gene knock-out experiments.
Synthetic sRNAs can be utilized for diverse experiments where gene expression regulation is needed. One of promising applications is high-throughput screening of the target genes to be manipulated and multiple strains simultaneously to enhance the production of chemicals and materials of interest. Such simultaneous optimization of gene targets and strains has been one of the big challenges in metabolic engineering. Another application is to fine tune the expression of the screened genes for flux optimization, which would enhance chemical production further by balancing the flux between biomass formation and target chemical production. Synthetic sRNAs can also be applied to finely regulating genetic interactions in a circuit or network, which is essential in synthetic biology. Once a sRNA scaffold-harboring plasmid is constructed, tailor-made, synthetic sRNAs can be made within 3-4 days, followed by the desired application experiments.
Dr. Eytan Zlotorynski, an editor at Nature Protocols, said "This paper describes the detailed protocol for the design and applications of synthetic sRNA. The method, which has many advantages, is likely to become common practice, and prove useful for metabolic engineering and synthetic biology studies."
This paper published in Nature Protocols will be useful for all researchers in academia and industry who are interested in the use of synthetic sRNAs for fundamental and applied biological and biotechnological studies.
This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) and the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Science, ICT and Future Planning through the National Research Foundation of Korea.
2013.10.31 View 8792