Scientists Re-engineer E. coli and unwittingly construct a case for a Creator
Runaway gas prices in recent years and the threat of climate change have prompted a number of scientists and technologists to search for cheap, renewable sources of energy to power automobiles.
Ethanol has been considered as a possible source of fuel, but its use has become controversial for a number of reasons, especially cost. Ethanol can’t serve as a total replacement for gasoline. It must be blended with petrol. Currently, corn is one of the chief sources of ethanol. Unfortunately, the cost of producing ethanol is more than the value of the energy it provides. Use of corn to produce ethanol also raises concerns about increased costs and a reduced supply of food. Some have suggested that switch grasses and celluloses may be a better option, but it remains to be determined if these raw materials are cost-effective sources of ethanol.
These problems have forced researchers to consider other options. One possible alternative is the use of alcohols with five carbons or more (C5 alcohols). C5 to C8 aliphatic alcohols are more similar to gasoline than ethanol (which is a C2 alcohol). These compounds are also much less soluble in water. This makes them easier to purify from water. (Ethanol has a high solubility in water.)
One approach to generate C5 to C8 aliphatic alcohols in an environmentally friendly and cost-effective way is to use microbes to produce these possible fuel compounds. Some bacteria have metabolic pathways that produce alcohols, but the compounds they generate are smaller than the desired C5 materials.
This term refers to the myriad chemical reactions that occur in organisms. These reactions are necessary to sustain life. Metabolic activity makes it possible for life-forms to extract energy from the environment and make life’s component parts.
Metabolic reactions include the production and breakdown of proteins and RNA molecules, DNA replication, and the assembly of cell membranes and cell walls. Metabolism also involves reactions of small molecules. For example, compounds like glucose and other sugar molecules are broken down into smaller molecules to provide energy for the cell’s operations. A significant number of metabolic reactions produce small molecules that are used by the cell’s machinery as building blocks to assemble proteins, DNA, the RNAs, and cell membrane bilayers. Some metabolic activities prepare materials the cell no longer needs–cellular waste–for elimination. Other reactions detoxify materials harmful to the cell.
Within the cell’s interior, metabolic processes are often organized into pathways comprised of a series of chemical reactions that transform a starting compound into a final product via a series of small, stepwise chemical changes. Each step in a metabolic route is mediated by a protein (called an enzyme) that assists in the chemical transformation. Metabolic pathways can be linear, branched, or circular. The chemical components that are part of a particular metabolic sequence sometimes take part in other metabolic pathways. These shared compounds cause the pathways to be interconnected and networked together.
Metabolic Engineering
Reengineering metabolic pathways to produce nonnatural materials like C5 alcohols is no easy task. One difficulty stems from the limited set of pathways and metabolites found in living systems. This doesn’t give biotechnologists many ways to use biochemistry to make non-natural materials. Still, bioengineers can take advantage of the available metabolic pathways to make some “artificial” materials by feeding nonnatural ingredients to cells. This approach has limitations though. Because of the specificity of the enzymes that catalyze metabolic routes, many nonnatural compounds won’t interact with the metabolic machinery and, hence, undergo chemical transformation into the desired product. For example, the Ehrlich Pathway is the only well-studied metabolic pathway that can form aliphatic alcohols. This route can be exploited to make alcohols by feeding microbes amino acids. However, it can generate only alcohols smaller than C5.
To overcome this limitation, researchers need to find a way to expand pathways, extending the chemistry and the range of metabolites that can be processed. This is an extremely challenging prospect. Metabolic pathways operate using the collective function of multiple enzymes that work sequentially in conjunction with one another. Generally speaking, to build upon the existing chemistry of a metabolic pathway, researchers have to reengineer the entire enzyme collective. Because of the complexity of metabolic pathways, bioengineers have to expend considerable effort on rational design strategies to achieve this reengineering, as the recent work on E. coli attests.
Reengineering E. coli to Produce C5–C8 Alcohols
To create a novel pathway in E.coli capable of generating alcohols larger than C5, the scientists relied on a common process to add carbon atoms to metabolites. Metabolic pathways like the tricarboxylic cycle, glyoxylate cycle, mevalonate pathway, and leucine biosynthesis use the compound acetyl-CoA as the source for carbon addition. They reasoned that they could take advantage of enzymes that used acetyl-CoA as a substrate) to make a C6 alcohol.
In this case, the researchers noted that the compound 2-keto-isovalerate can be converted to the larger compound 2-ketoisocaproate through a three-step elongation pathway that relies on acetyl- CoA. This reaction sequence is catalyzed by the enzymes dubbed LeuA, LeuC, LeuD, and LeuB (2-isopropylmalate synthase, isopropylmalate isomerase complex, and 3-isopropylmalate dehydrogenase, respectively). The team determined that these enzymes could be used to generate 2-keto-4-methylhexanoate from 2-ketoisovalerate. Once they had formed 2-keto-4-methylhexanoate, the researchers reasoned that this compound could be converted to 3-methyl-1-pentanol (a C6 alcohol) by the sequential action of the enzymes KVID (2-ketoisovalerate decarboxylase) from the bacterium Lactococcus lactis and ADH6 (alcohol dehydrogenase) from the yeast Saccharomyces cerevisiae. To make the starting material, 2-keto-isovalerate, they speculated that they could use the intrinsic metabolic capacity of E. coli to make the related compound 2-keto-3-methylvalerate.
With their strategy in place, the biochemists redesigned some of the key enzymes in the nonnatural metabolic pathway they constructed. They used structural data for the enzyme and substrates along with detailed knowledge of the reaction mechanisms to strategically replace amino acids in the binding and active sites of KVID and LeuA. This was done so that the re-engineered enzymes would operate preferentially on the nonnatural metabolic intermediates 2-keto-4-methylhexanoate and 2-ketoisovalerate, respectively.
Once they had mapped out the novel metabolic pathway on paper and had produced reengineered versions of LeuA and KVID, the researchers went into the lab to introduce the newly devised capability to make C6 alcohols into E. coli. The team constructed three synthetic plasmids (circular pieces of DNA that can harbor genes and be taken up by bacteria) that contained sets of genes with the information to make enzymes involved in the production of C5 to C8 alcohols. Two of the plasmids were comprised of genes that were already present in the E. coli genome. The other plasmid contained novel genes that encoded the proteins LeuA, LeuB, LeuC, LeuD, KVID, and ADH6. The presence of the extra genes in the bacterium resulted in an overproduction of the enzymes needed to generate key metabolites in the production of the C6 alcohol. By introducing two plasmids with genes already found in E. coli, the researchers were able to ensure that most of the foodstuff (glucose) consumed by the bacteria would be metabolized to produce the C6 alcohol.
The plasmids were also designed to include a biochemical on/off switch of sorts (called a promoter). This on/off control allowed the researchers to “turn on” the plasmid genes by feeding the bacteria with a specific sugar (lactose).
Using this approach, the scientists were able to successfully produce relatively high levels of the C6 alcohol. This work opens up the opportunity to use bioengineering of metabolic pathways in microbes as a way to generate alternative sources of energy. It also provides empirical evidence that life must stem from the work of a Creator. Here’s how.
Reengineering Metabolism and the Case for Intelligent Design
If not for their ingenuity and strategic efforts based on decades of accumulated knowledge and insight, the researchers would never have been able to design a novel, nonnatural metabolic pathway capable of generating C5 to C8 alcohols. They very carefully mapped out a plausible pathway relying on understanding of the metabolic capabilities of living systems and redesigned key enzymes from a variety of sources using a detailed understanding of the chemistry involved to engineer a new pathway. Then, the team pain-stakingly selected the appropriate microorganism to harbor the non-natural metabolic route they designed. Finally, they cleverly conceived and constructed the plasmids to transfer the necessary genes into E. coli and to regulate their activity in such a way that the production of the C6 alcohol was favored.
It is worth noting–as marvelous as this achievement is–that the researchers didn’t create this metabolic pathway from scratch, but pieced together the pathway using modified enzymes taken from a variety of sources.
In the end, it’s fair to say that this novel metabolic process was intelligently designed. In fact, biochemists describe this type of work as rational design. Given the amount of effort invested in reengineering existing metabolic systems to make C5 to C8 alcohols, the design of this artificial metabolic system 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 intelligent agency in order to come into being and to undergo significant change?
Such outstanding work holds promise to help solve energy problems and also fuels the case for intelligent design.
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