A few days ago while fueling up my car I was “elated” (and by elated, I mean annoyed) to discover that the price of gasoline was over $3.00 a gallon—again. Recent work by a team of synthetic biologists from UC Berkeley makes me optimistic, however, that high gas prices may soon be an inconvenience of the past.
These scientists engineered the bacterium E. coli to produce biodiesel from hemicelluloses, a component of plant waste.1 Their work represents an important milestone in commercializing a clean, renewable form of energy that holds the potential to generate 85% less greenhouse gases than standard diesel.2
It also adds to the evidence for biochemical intelligent Design.
Crude oil is not the only source of diesel. Cells possess the metabolic pathways to make their own diesel fuel, of sorts: fatty acids. Diesel fuel consists mostly of high molecular weight hydrocarbons. In a similar vein, fatty acids are comprised of long hydrocarbon chains. Scientists can extract fatty acids and resulting biochemical derivatives from animal and plants sources for use as biodiesel and as starting materials to make surfactants, solvents, and lubricants.
There are drawbacks, however, with using animals and plants as a source for biodiesel. These sources can provide only a limited supply, which is already being outpaced by growing demand. Questionable land practices and environmental concerns associated with the production of biodiesel further compound this problem. Biotechnologists hope engineering microbes to produce biodiesel will address these limitations. E. coli is particularly well-suited for this use because it consists of about 10% fat and can produce fatty acids at a commercially viable rate. Furthermore, these microbes are also amenable to genetic engineering.
The synthetic biologists from UC Berkeley performed over a dozen genetic modifications to the E. coli genome in order to generate a nonnatural strain capable of producing fatty acids, fatty esters, fatty alcohols, and waxes at artificially high levels. As part of these bioengineering efforts, they introduced foreign genes into E. coli’s genome to impart this microbe with the capacity to digest hemicelluloses into simple sugars. The researchers also amplified and commandeered intrinsic metabolic pathways to produce fatty acids at high levels and at the same time shut down other processes that normally redirect metabolites away from fatty acid biosynthesis or inhibit it altogether.
This strategy seems simple and straightforward on paper. In reality, its detailed execution requires thoughtful planning and skilled laboratory manipulations by the research team—it represents science at its very best. To appreciate their efforts, it’s instructive to consider what it took for these scientists to successfully transform a wild-type strain of E. coli into one that produces biodiesel.
In E. coli, the end product of fatty acid biosynthesis is a biomolecular complex called fatty acid-ACP. This complex consists of a protein (called acyl carrier protein or ACP) with a fatty acid joined to it via a special type of chemical linkage called a thioester bond. The accumulation of fatty acid-ACP inside the cell inhibits the metabolic pathways that produce fatty acids. This feedback inhibition serves as a regulatory device to keep the cell from overproducing fatty acids.
In order to produce fatty acids at high enough levels to support commercial applications, the researchers realized they had to deregulate fatty acid biosynthesis. To do this they altered the gene that encodes an enzyme known as a thioesterase, thereby causing the protein to accumulate in the cytoplasm of the cell (as opposed to its natural gathering place in the periplasmic space between the inner and outer membranes). This enzyme breaks down the fatty acid-ACP complex by cleaving the thioester linkage between the fatty acid and the protein. The investigators learned this destruction short-circuited the feedback regulation of fatty acid biosynthesis, thus, leading to the nonnatural, high-level accumulation of fatty acids in the cytoplasm.
To further increase the accumulation, the researchers also shut down metabolic pathways that break down fatty acids (called β-oxidation). They did this by deactivating two genes that specify enzymes that initiate the first steps of fatty acid breakdown.
Fatty acids are valuable raw materials, but to be used as biodiesel they must be converted into compounds known as fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). This conversion can be done on an industrial scale by isolating the fatty acids from E. coli and then causing them to react with either methyl alcohol or ethyl alcohol.
However, the UC Berkeley researchers wanted to avoid this step and engineer E. coli to produce FAEEs directly. To achieve this, they added genes from the bacterium Zymomonas mobilis (a microbe known for producing ethanol). These genes encode the enzymes pyruvate decarboxylase and alcohol dehydrogenase, which operate sequentially to convert pyruvate into ethyl alcohol. (Pyruvate is a common metabolite generated when the cell’s metabolic pathways break down sugars.) Once produced, ethyl alcohol can then react with the fatty acids to generate biodiesel. Enzymes called acyltransferases mediate the reaction. With these modifications in place (and when fed sugar), E. coli can make biodiesel.
Yet again, the researchers wanted to take things a step further. They aimed to develop a strain of E. coli capable of generating biodiesel from plant waste. To that end, the team added two genes to the E. coli genome: one (endoxylanase) from the microbe Clostridium stercorarium and another (xylanase) from Bacteroides ovatus. The combined action of these two enzymes results in the breakdown of hemicelluloses into the sugar xylose that E. coli can use to make biodiesel. And since hemicellulose is insoluble in the growth medium, the researchers designed E. coli so that when it produced endoxylanase and xylanase, the two enzymes would be fused to the protein OsmY located in the E. coli outer membrane and, thus, be allowed access to hemicellulose.
The Production of Biodiesel and the Case for Intelligent Design
As I pointed out in a previous article, the researchers would never have been able to engineer E. coli to make biodiesel if not for their ingenuity and strategic planning based on decades of accumulated knowledge and insight. It’s worth noting—as marvelous as this achievement is—that the UC Berkeley scientists didn’t create this metabolic capability from scratch. Rather they pieced together the pathway using modified enzymes taken from a variety of sources and by manipulating the activity of endogenous E. coli genes.
In the end, it’s fair to say this novel metabolic process was intelligently designed. In fact, biochemists describe this type of work as rational design. The amount of effort invested in reengineering existing metabolic systems to make biodiesel raises provocative questions. Is it reasonable to maintain that life’s chemistry originated and evolved through undirected processes? Doesn’t this work provide direct, empirical evidence that biochemical systems require the work of an intelligent agency in order to come into being and to undergo significant change?
1. Eric J. Steen et al., “Microbial Production of Fatty-Acid-Derived Fuels and Chemicals from Plant Biomass,” Nature 463 (2010): 559–63.
2. Jeff Tollefson, “Altered Microbe Makes Biofuel,” Nature 463 (2010): 409.