Engineers Reverse E. Coli Metabolism for Quick Production of Fuels, Chemicals

Engineers Reverse E. Coli Metabolism for Quick Production of Fuels, Chemicals

  10:54:00 PM    Science Lover

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In a biotechnological tour de force, Rice University engineering researchers this week unveiled a new method for rapidly converting simple glucose into biofuels and petrochemical substitutes. In a paper published online in Nature, Rice's team described how it reversed one of the most efficient of all metabolic pathways -- the beta oxidation cycle -- to engineer bacteria that produce biofuel at a breakneck pace.

Rice University engineering researchers Ramon Gonzalez (left) and
Clementina Dellomonaco reversed one of the most efficient of all
metabolic pathways -- the beta oxidation cycle -- to engineer bacteria
that make biofuels at a breakneck pace. (Credit: Jeff Fitlow/Rice
University)

Just how fast are Rice's single-celled chemical factories? On a cell-per-cell basis, the bacteria produced the butanol, a biofuel that can be substituted for gasoline in most engines, about 10 times faster than any previously reported organism.


"That's really not even a fair comparison because the other organisms used an expensive, enriched feedstock, and we used the cheapest thing you can imagine, just glucose and mineral salts," said Ramon Gonzalez, associate professor of chemical and biomolecular engineering at Rice and lead co-author of the Nature study.


Gonzalez's laboratory is in a race with hundreds of labs around the world to find green methods for producing chemicals like butanol that have historically come from petroleum.


"We call these 'drop-in' fuels and chemicals, because their structure and properties are very similar, sometimes identical, to petroleum-based products," he said. "That means they can be 'dropped in,' or substituted, for products that are produced today by the petrochemical industry."




Butanol is a relatively short molecule, with a backbone of just four carbon atoms. Molecules with longer carbon chains have been even more troublesome for biotech producers to make, particularly molecules with chains of 10 or more carbon atoms. Gonzalez said that's partly because researchers have focused on ramping up the natural metabolic processes that cells use to build long-chain fatty acids. Gonzalez and students Clementina Dellomonaco, James Clomburg and Elliot Miller took a completely different approach.


"Rather than going with the process nature uses to build fatty acids, we reversed the process that it uses to break them apart," Gonzalez said. "It's definitely unconventional, but it makes sense because the routes nature has selected to build fatty acids are very inefficient compared with the reversal of the route it uses to break them apart."


The beta oxidation process is one of biology's most fundamental, Gonzalez said. Species ranging from single-celled bacteria to human beings use beta oxidation to break down fatty acids and generate energy.


In the Nature study, Gonzalez's team reversed the beta oxidation cycle by selectively manipulating about a dozen genes in the bacteria Escherichia coli. They also showed that selective manipulations of particular genes could be used to produce fatty acids of particular lengths, including long-chain molecules like stearic acid and palmitic acid, which have chains of more than a dozen carbon atoms.


"This is not a one-trick pony," Gonzalez said. "We can make many kinds of specialized molecules for many different markets. We can also do this in any organism. Some producers prefer to use industrial organisms other than E. coli, like algae or yeast. That's another advantage of using reverse-beta oxidation, because the pathway is present in almost every organism."


The research was funded by Rice University.

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Climate Change Reducing Ocean's Carbon Dioxide Uptake, New Analysis Shows

Climate Change Reducing Ocean's Carbon Dioxide Uptake, New Analysis Shows

  9:52:00 AM    Science Lover

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How deep is the ocean's capacity to buffer against climate change?

A new analysis provides some of the 
first observational evidence that 
climate change is negatively 
impacting the ocean carbon sink. 
(Credit: © lunamarina / Fotolia)

As one of the planet's largest single carbon absorbers, the ocean takes up roughly one-third of all human carbon emissions, reducing atmospheric carbon dioxide and its associated global changes.

But whether the ocean can continue mopping up human-produced carbon at the same rate is still up in the air. Previous studies on the topic have yielded conflicting results, says University of Wisconsin-Madison assistant professor Galen McKinley.

In a new analysis published online July 10 in Nature Geoscience, McKinley and her colleagues identify a likely source of many of those inconsistencies and provide some of the first observational evidence that climate change is negatively impacting the ocean carbon sink.

"The ocean is taking up less carbon because of the warming caused by the carbon in the atmosphere," says McKinley, an assistant professor of atmospheric and oceanic sciences and a member of the Center for Climatic Research in the Nelson Institute for Environmental Studies.

The analysis differs from previous studies in its scope across both time and space. One of the biggest challenges in asking how climate is affecting the ocean is simply a lack of data, McKinley says, with available information clustered along shipping lanes and other areas where scientists can take advantage of existing boat traffic. With a dearth of other sampling sites, many studies have simply extrapolated trends from limited areas to broader swaths of the ocean.

McKinley and colleagues at UW-Madison, the Lamont-Doherty Earth Observatory at Columbia University, and the Universite Pierre et Marie Curie in Paris expanded their analysis by combining existing data from a range of years (1981-2009), methodologies, and locations spanning most of the North Atlantic into a single time series for each of three large regions called gyres, defined by distinct physical and biological characteristics.

They found a high degree of natural variability that often masked longer-term patterns of change and could explain why previous conclusions have disagreed. They discovered that apparent trends in ocean carbon uptake are highly dependent on exactly when and where you look -- on the 10- to 15-year time scale, even overlapping time intervals sometimes suggested opposite effects.



"Because the ocean is so variable, we need at least 25 years' worth of data to really see the effect of carbon accumulation in the atmosphere," she says. "This is a big issue in many branches of climate science -- what is natural variability, and what is climate change?"

Working with nearly three decades of data, the researchers were able to cut through the variability and identify underlying trends in the surface CO2 throughout the North Atlantic.

During the past three decades, increases in atmospheric carbon dioxide have largely been matched by corresponding increases in dissolved carbon dioxide in the seawater. The gases equilibrate across the air-water interface, influenced by how much carbon is in the atmosphere and the ocean and how much carbon dioxide the water is able to hold as determined by its water chemistry.

But the researchers found that rising temperatures are slowing the carbon absorption across a large portion of the subtropical North Atlantic. Warmer water cannot hold as much carbon dioxide, so the ocean's carbon capacity is decreasing as it warms.

In watching for effects of increasing atmospheric carbon on the ocean's uptake, many people have looked for indications that the carbon content of the ocean is rising faster than that of the atmosphere, McKinley says. However, their new results show that the ocean sink could be weakening even without that visible sign.

"More likely what we're going to see is that the ocean will keep its equilibration but it doesn't have to take up as much carbon to do it because it's getting warmer at the same time," she says. "We are already seeing this in the North Atlantic subtropical gyre, and this is some of the first evidence for climate damping the ocean's ability to take up carbon from the atmosphere."

She stresses the need to improve available datasets and expand this type of analysis to other oceans, which are relatively less-studied than the North Atlantic, to continue to refine carbon uptake trends in different ocean regions. This information will be critical for decision-making, since any decrease in ocean uptake may require greater human efforts to control carbon dioxide levels in the atmosphere.

McKinley's work on the project was supported by the National Aeronautics and Space Administration.

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LCD Projector Used to Control Brain and Muscles of Tiny Organisms Such as Worms

LCD Projector Used to Control Brain and Muscles of Tiny Organisms Such as Worms

  10:22:00 AM    Science Lover

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Researchers are using inexpensive components from ordinary liquid crystal display (LCD) projectors to control the brain and muscles of tiny organisms, including freely moving worms. Red, green and blue lights from a projector activate light-sensitive microbial proteins that are genetically engineered into the worms, allowing the researchers to switch neurons on and off like light bulbs and turn muscles on and off like engines.

Hang Lu, an associate professor in the School of Chemical 
& Biomolecular Engineering at Georgia Tech, and her 
graduate students Jeffrey Stirman (left) and Matthew Crane 
are using inexpensive LCD projectors to control the brain 
and muscles of tiny organisms, including freely moving 
worms. (Credit: Georgia Tech/Gary Meek)

Use of the LCD technology to control small animals advances the field of optogenetics -- a mix of optical and genetic techniques that has given researchers unparalleled control over brain circuits in laboratory animals. Until now, the technique could be used only with larger animals by placement of an optical fiber into an animal's brain, or required illumination of an animal's entire body.

A paper published Jan. 9 in the advance online edition of the journal Nature Methods describes how the inexpensive illumination technology allows researchers to stimulate and silence specific neurons and muscles of freely moving worms, while precisely controlling the location, duration, frequency and intensity of the light.

"This illumination instrument significantly enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals," said Hang Lu, an associate professor in the School of Chemical & Biomolecular Engineering at the Georgia Institute of Technology.

Lu and graduate students Jeffrey Stirman and Matthew Crane developed the tool with support from the National Institutes of Health and the Alfred P. Sloan Foundation.

The illumination system includes a modified off-the-shelf LCD projector, which is used to cast a multi-color pattern of light onto an animal. The independent red, green and blue channels allow researchers to activate excitable cells sensitive to specific colors, while simultaneously silencing others.

"Because the central component of the illumination system is a commercially available projector, the system's cost and complexity are dramatically reduced, which we hope will enable wider adoption of this tool by the research community," explained Lu.

By connecting the illumination system to a microscope and combining it with video tracking, the researchers are able to track and record the behavior of freely moving animals, while maintaining the lighting in the intended anatomical position. When the animal moves, changes to the light's location, intensity and color can be updated in less than 40 milliseconds.

Once Lu and her team built the prototype system, they used it to explore the "touch" circuit of the worm Caenorhabditis elegans by exciting and inhibiting its mechano-sensory and locomotion neurons. Alexander Gottschalk, a professor in the Johann Wolfgang Goethe-University Frankfurt Institute of Biochemistry in Frankfurt, Germany, and his team provided the light-sensitive optogenetic reagents for the Georgia Tech experiments.

For their first experiment, the researchers illuminated the head of a worm at regular intervals while the animal moved forward. This produced a coiling effect in the head and caused the worm to crawl in a triangular pattern. In another experiment, the team scanned light along the bodies of worms from head to tail, which resulted in backward movement when neurons near the head were stimulated and forward movement when neurons near the tail were stimulated.

Additional experiments showed that the intensity of the light affected a worm's behavior and that several optogenetic reagents excited at different wavelengths could be combined in one experiment to understand circuit functions. The researchers were able to examine a large number of animals under a variety of conditions, demonstrating that the technique's results were both robust and repeatable.

"This instrument allowed us to control defined events in defined locations at defined times in an intact biological system, allowing us to dissect animal functional circuits with greater precision and nuance," added Lu.

While these proof-of-concept studies investigated the response of C. elegans to mechanical stimulation, the illumination system can also be used to evaluate responses to chemical, thermal and visual stimuli. Researchers can also use it to study a variety of neurons and muscles in other small animals, such as the zebrafish and fruit fly larvae.

"Experiments with this illumination system yield quantitative behavior data that cannot be obtained by manual touch assays, laser cell ablation, or genetic manipulation of neurotransmitters," said Lu.

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