E. coli bacteria engineered to eat switchgrass and make transportation fuels

 A milestone has been reached on the road to developing advanced biofuels that can replace gasoline, diesel and jet fuels with a domestically-produced clean, green, renewable alternative.

Researchers with the U.S. Department of Energy (DOE)'s Joint BioEnergy Institute (JBEI) have engineered the first strains of Escherichia colibacteria that can digest switchgrass biomass and synthesize its sugars into all three of those transportation fuels. What's more, the microbes are able to do this without any help from enzyme additives.

"This work shows that we can reduce one of the most expensive parts of the biofuel production process, the addition of enzymes to depolymerize cellulose and hemicellulose into fermentable sugars," says Jay Keasling, CEO of JBEI and leader of this research. "This will enable us to reduce fuel production costs by consolidating two steps -- depolymerizing cellulose and hemicellulose into sugars, and fermenting the sugars into fuels -- into a single step or one pot operation."

Keasling, who also holds appointments with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkley, is the corresponding author of a paper in the Proceedings of the National Academy of Sciences (PNAS) that describes this work. The paper is titled "Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli."

Advanced biofuels made from the lignocellulosic biomass of non-food crops and agricultural waste are widely believed to represent the best source of renewable liquid transportation fuels. Unlike ethanol, which in this country is produced from corn starch, these advanced biofuels can replace gasoline on a gallon-for-gallon basis, and they can be used in today's engines and infrastructures. The biggest roadblock to an advanced biofuels highway is bringing the cost of producing these fuels down so that they are economically competitive.

Unlike the simple sugars in corn grain, the cellulose and hemicellulose in plant biomass are difficult to extract in part because they are embedded in a tough woody material called lignin. Once extracted, these complex sugars must first be converted or hydrolyzed into simple sugars and then synthesized into fuels. At JBEI, a DOE Bioenergy Research Center led by Berkeley Lab, one approach has been to pre-treat the biomass with an ionic liquid (molten salt) to dissolve it, then engineer a single microorganism that can both digest the dissolved biomass and produce hydrocarbons that have the properties of petrochemical fuels.

"Our goal has been to put as much chemistry as we can into microbes," Keasling says. "For advanced biofuels this requires a microbe with pathways for hydrocarbon production and the biomass-degrading capacity to secrete enzymes that efficiently hydrolyze cellulose and hemicellulose. We've now been able to engineer strains of Escherichia coli that can utilize both the cellulose and hemicellulose fractions of switchgrass that's been pre-treated with ionic liquids."

E. coli bacteria normally cannot grow on switchgrass, but JBEI researchers engineered strains of the bacteria to express several enzymes that enable them to digest cellulose and hemicellulose and use one or the other for growth. These cellulolytic and hemicellulolytic strains of E. coli, which can be combined as co-cultures on a sample of switchgrass, were further engineered with three metabolic pathways that enabled the E. coli to produce fuel substitute or precursor molecules suitable for gasoline, diesel and jet engines. While this is not the first demonstration of E. coli producing gasoline and diesel from sugars, it is the first demonstration of E. coli producing all three forms of transportation fuels. Furthermore, it was done using switchgrass, which is among the most highly touted of the potential feedstocks for advanced biofuels.

Gregory Bokinsky, a post-doctoral researcher with JBEI's synthetic biology group and lead author of the PNAS paper, explains that the pre-treatment of the switchgrass with ionic liquids was essential to this demonstration.

"The magic is in the ionic liquid pre-treatment," Bokinsky says. "If properly optimized, I suspect you could use ionic liquid pre-treatment on any plant biomass and make it readily digestible by microbes. For us it was the combination of biomass from the ionic liquid pretreatment with the engineered E. coli that enabled our success."

The JBEI researchers also attribute the success of this work to the "unparalleled genetic and metabolic tractability" of E. coli, which over the years has been engineered to produce a wide range of chemical products. However, the researchers believe that the techniques used in this demonstration should also be readily adapted to other microbes. This would open the door to the production of advanced biofuels from lignocellulosic feedstocks that are ecologically and economically appropriate to grow and harvest anywhere in the world. For the JBEI researchers, however, the next step is to increase the yields of the fuels they can synthesize from switchgrass.

"We already have hydrocarbon fuel production pathways that give far better yields than what we obtained with this demonstration," says Bokinsky. "And these other pathways are very likely to be compatible with the biomass-consumption pathways we've engineered into our E. coli. However, we need to find enzymes that can both digest more of the ionic liquid pre-treated biomass and be secreted by E coli. We also need to work on optimizing the ionic liquid pre-treatment steps to yield biomass that is even easier for the microbes to digest."

Co-authoring the PNAS paper with Keasling and Bokinsky were Pamela Peralta-Yahya, Anthe George, Bradley Holmes, Eric Steen, Jeffrey Dietrich, Taek Soon Lee, Danielle Tullman-Ercek, Christopher Voigt and Blake Simmons.

This research was supported in part by the DOE Office of Science and a UC Discovery Grant.

Story Source:

The above story is reprinted from materials provided by DOE/Lawrence Berkeley National Laboratory.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Journal Reference:

Gregory Bokinsky, Pamela P. Peralta-Yahya, Anthe George, Bradley M. Holmes, Eric J. Steen, Jeffrey Dietrich, Taek Soon Lee, Danielle Tullman-Ercek, Christopher A. Voigt, Blake A. Simmons, Jay D. Keasling. Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proceedings of the National Academy of Sciences, 2011; DOI: 10.1073/pnas.1106958108

Emerging new properties at oxide interfaces

 Dr. Ariando of the National University of Singapore discovered a collective electronic state not seen before in the bulk of either two individual insulating oxides, thus demonstrating that electrons at their interface can now exhibit ferromagnetism.

In many ionic materials, including the oxides, surfaces created along specific directions can become electrically charged. By the same token, such electronic charging, or 'polarisation', can also occur at the interface of two connecting materials.

Theoretically, this could lead to the build-up of an ever increasing voltage in the materials in certain systems, a situation known as a 'polarity catastrophe'. Certainly this cannot occur in practical systems, for energy sake, and Nature deals with this situation by reconstructing the electronic configuration of the interface via a shifting of charges across the interface, or by structural reconstructions, namely, the displacement of atoms.

With oxide materials, a unique consequence of these reconstructions is that it provides a means to create novel electronic phases, stabilised by the interface, and which cannot exist in the bulk.

Dr. Ariando from the National University of Singapore's (NUS) Department of Physics and NUS Nanoscience and Nanotechnology-NanoCore, along with his co-workers, showed that at this interface, a remarkable combination of strong diamagnetism (superconductor like), paramagnetism and ferromagnetism can co-exist with the quasi two-dimensional electron gas when prepared under a more oxidising condition.

Past studies had shown that two-dimensional conducting planes, in the form of quasi two-dimensional electron gas, could emerge between otherwise non-magnetic insulating oxide, Lanthanum Alumniate (LaAlO3) and Strontium Titanate (SrTiO3).

Interestingly, Dr. Ariando's team had also shown that the ferromagnetic phase was stable even above room temperature and the diamagnetism below a relatively high temperature of 60 K.

Industrial applications

The results also indicate that the free surface of SrTiO3 could well be responsible for all these fascinating phenomena. The SrTiO3 resembles Silicon. This will have a significant impact on industry since Silicon has been used in semiconductor technology -- silicon has been the workhorse for oxide-based devices and electronics.

These multiple electronic and magnetic phases at oxide interfaces could yield interesting technological applications. That a variety of magnetic states can be produced close to the surface (< 10 nm) by changing the external stimulus to the SrTiO3 or the interface of LaAlO3/SrTiO3, be it change in oxygen pressure or magnetic field, thus proves that this is a very active interface, and that it can yield strong responses to external stimuli.

One could well consider building novel sensors out of these interfaces that could be used as, say, oxygen sensors, or even magnetic sensors. Still, where these applications are concerned, there is a need to further understand these phenomena and optimise the device configuration.

The research of Dr. Ariando and his co-workers in the oxide interface field is reminiscent of the times when two-dimensional electron gas in the semiconductor heterostructures first became available, and the quantum Hall effect and fractional quantum Hall effect were discovered, both resulting in Nobel prizes.

The physics of the oxide material systems is however richer, involving much stronger interaction between the electrons, mutually and within the crystal lattice. There is great interest in exploring these interfaces in the quest for new nano-electronic devices.

Story Source:

The above story is reprinted from materials provided by National University of Singapore, via AlphaGalileo.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Journal Reference:

Ariando, X. Wang, G. Baskaran, Z. Q. Liu, J. Huijben, J. B. Yi, A. Annadi, A. Roy Barman, A. Rusydi, S. Dhar, Y. P. Feng, J. Ding, H. Hilgenkamp, T. Venkatesan. Electronic phase separation at the LaAlO3/SrTiO3 interface. Nature Communications, 2011; 2: 188 DOI: 10.1038/ncomms1192

Tuberculosis researchers discover potential new target for treatments

The enzyme is an especially important discovery because it is present in both replicating and non-replicating strains of the bacteria, including resistant strains. That’s key because non-replicating bacteria are much more difficult to kill with antibiotics, which is one reason treatments for tuberculosis are so long-lasting.

“Another interesting observation that arose from our work is that this enzyme – known as FBA-tb – is on the surface of the bacterium. Because it is on the surface, it has the ability to interact with a human substance called plasminogen, which plays a key role in our immune response,” said Mary Jackson, an associate professor in CSU’s Department of Microbiology, Immunology and Pathology. Jackson works in the Mycobacteria Research Laboratories. “This finding suggests that the bacteria that cause tuberculosis may use this enzyme to manipulate our immune system and spread tuberculosis throughout our body. We’re looking into that theory now.”

Jackson’s laboratory also is already pursuing research to find ways to block this enzyme from helping the bacteria that causes tuberculosis replicate. That research is being funded by two National Institutes of Health grants.

The research discovery is published in today’s issue of the Journal of Biological Chemistry.

Tuberculosis is one of the world’s deadliest diseases, with one-third of the entire global population being infected. In 2010, about 1.4 million people died of tuberculosis or illnesses related to the disease, and 9 million people became ill with tuberculosis, according to the Centers for Disease Control. While tuberculosis is more common in other countries, the CDC says more than 11,000 new cases were reported in the United States in 2010.

Provided by Colorado State University

Researchers show how iron activates oxygen in living things

The team used high-powered X-rays from the Stanford Synchrotron Radiation Lightsource to capture the fine details of how these enzymes work. The results could have applications in medicine, energy production and industrial processes.

Led by Edward Solomon, a professor of photon science at SLAC, Wonwoo Nam, of Ewha Womans University in South Korea, and Joan Valentine of the University of California, Los Angeles, the team reported its findings in the Oct. 27 issue of Nature.

“It’s what we’re all after: How does nature make these metal sites do this chemistry better than scientists can do in the labs?” Solomon said.

In its most abundant form, oxygen exists as a two-atom molecule, O2. Electronically speaking, O2 is “forbidden” from reacting with other biological molecules until it is split into two separate oxygen atoms.

Specialized enzymes, containing metallic elements like iron, drive this important preparatory step. The precise mechanism of oxygen activation by iron complexes has long eluded researchers, in part because the reaction—which proceeds through multiple intermediate stages—happens in mere fractions of a second.

Researchers recently captured all three of the intermediate structures that one iron complex morphs into as it cleaves the O2 bond – including one wily intermediate that exists for less than 2 milliseconds before converting into a different form.

They used two specialized instruments at the SSRL to determine the electronic and geometric structure of each intermediate stage. Chemical tests at Ewha Womans University further revealed that these iron-based intermediates are versatile chemical catalysts, able to react with both electron-rich and electron-deficient molecules.

This is the first time researchers have so fully characterized this type of iron- and oxygen-containing molecule — called non-heme iron because it lacks the heme group for which the iron-containing molecule hemoglobin that carries oxygen in red blood cells is named.

Solomon says the newly discovered reaction mechanism could help scientists better understand diseases of non-heme iron enzymes, such as phenylketonuria. The disease, which prevents breakdown of the amino acid phenylalanine, can cause developmental defects in babies and lasting health problems for adults.

Other more distant applications may affect energy production and industrial processes that use similar chemistry. One day, Solomon says, industrial chemists may find a way to co-opt the non-heme iron enzyme reaction to drive reactions more quickly and cheaply.

Other co-authors on the paper, "Structure and reactivity of a mononuclear non-haem iron(III)–peroxo complex," include Stanford photon science professors Britt Hedman, who is also deputy director of SSRL, and Keith Hodgson, SLAC's chief research officer.

Provided by SLAC National Accelerator Laboratory (news : web)

Research group develops more efficient artificial enzyme

Though the artificial enzyme is still many orders of magnitude less efficient than nature’s way of doing things, it is far more efficient than any other artificial process to date, a milestone that gives researchers hope that they will one day equal nature’s abilities.

The mimic, as the team refers to it, is made up of a three-stranded “coiled coil” protein that binds two metals: the active site is zinc, sitting inside the hydrophobic center, while the other site is made up of mercury metals that help to keep the whole works stabilized when introduced to a high pH environment.

The team says that their metalloprotein isn’t necessarily an end product, but more of a way to prove that there likely does exist a path to creating artificial enzymes that can be every bit as efficient as those that occur in nature. The whole point being that if artificial ones can be created, they could be mass produced to exact specifications and used in large scale ways, such as sequestering carbon dioxide in the atmosphere and turning it to a harmless substance that would fall to the ground as bicarbonate.

The next step in the research is to copy the so-called “'second sphere” part of natural enzymes that is missing from the artificial mimic. Its role is to stabilize the differing states during transition and to aid in transferring protons.

It’s because the mimic was able to beat current efficiency models without the second sphere that the researchers are so optimistic about further improvements to their artificial enzyme. Figuring out how to add that second sphere may help the team gain a 100 to 500 fold increase in efficiency of their metalloprotein, which would make it nearly as efficient as Mother Nature, a feat that might yet lead to not only a tool to help combat global warming, but to all manner of applications in the medical field.

More information: Hydrolytic catalysis and structural stabilization in a designed metalloprotein, Nature Chemistry (2011) doi:10.1038/nchem.1201

Abstract
Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.

Project page: http://www.umich.e … peptide.html