Novel technology to produce gasoline by a metabolically-engineered microorganism

Sep. 29, 2013 — Scientists succeeded in producing 580 mg of gasoline per litre of cultured broth by converting in vivo generated fatty acid.

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 titre 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 titre, 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."

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Plants in space: A novel method for fixing plant tissue samples maximizes time, resources, and data

Sep. 11, 2013 — At work on the International Space Station, researchers studying plant and cell growth in space encountered a challenge. Imaging revealed interesting spaceflight-associated root morphologies. They needed to fix the tissues for further study back on Earth, but conventional fixation methods require separate fixatives depending on whether the sample is intended for molecular or morphological study. If the scientists wanted to study how spaceflight affected patterns of gene expression central to morphological patterns of cell growth, they needed a fixation method that would allow them to study both perspectives.

Most scientists at work in the laboratory rely on protocols that have been developed without the need for restrictions on the amount of space, time, or reagents they use. For scientists conducting experiments in spaceflight, time and resources are strictly regulated and limited, and researchers must know in advance which protocols will maximize the usefulness of the data they collect.

University of Florida professors Anna-Lisa Paul and Robert Ferl and colleagues are collaborating with the National Aeronautics and Space Administration (NASA) to understand plant growth and development in spaceflight. Along with lead author and graduate student Eric Schultz, they have developed a single fixation protocol for use in space that allows plant material to be used for multiple experimental applications. Their new protocol for sample preparation was tested on Arabidopsis tissues harvested on the International Space Station and is described in the August issue of Applications in Plant Sciences.

Because of limitations in astronaut crew time and orbital resources, previous spaceflight fixation protocols were designated as either molecular or morphological, due to the separate fixatives required for each application. Tissues for morphologic study were fixed in 3% glutaraldehyde (or a similar solution), and tissues for molecular study were fixed in the tissue storage reagent RNAlater. RNAlater has not commonly been used as a morphologic fixative, as it can produce unclear images with high background staining.

The new method developed by Schultz et al. puts RNAlater-fixed samples through a desalination process to return them to a fresh-like state, and then uses low-temperature scanning electron microscopy (cryo-SEM) to preserve tissues for imaging. Because few laboratories have access to the necessary equipment for cryo-SEM, the authors tested and developed a protocol that emulates cryo-SEM using standard SEM equipment and, importantly, that results in minimal tissue damage.

Although it was developed to address specific constraints for spaceflight experiments, Paul notes that their new method is broadly applicable. "There are a lot of situations where biologists want to collect samples in extreme situations. In our case -- a space vehicle orbiting the Earth."

The new protocol maximizes the amount of data obtained from a single sample and allows for the concomitant examination of both molecular and morphological features. Using a single fixation protocol, direct comparisons between changes in morphology and altered gene expression can be made. Such an analysis not only makes full use of samples and replicates but also enables a robust analysis of the relationship between heredity and development. "Putting two tools together, it is powerful to look at the morphology in conjunction with the genes that are being expressed," says Paul.

The new protocol boasts low costs and high accessibility, and has wide application to any situation where recovery of biological resources is limited. Notably, this includes researchers collecting and preserving samples in the field, where space for materials is at a premium. "In places where sampling is limited, difficult, or expensive, the use of preservatives allows for more routes to analysis," notes Ferl.

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