New Pathways Could Improve Ethanol Yields

A close look reveals metabolic conversion of oil content into sugar is possible
By Trent Nguyen, Ph.D. | February 22, 2012

With the current demand for energy, fossil fuel is estimated to be depleted within 60 to 80 years and energy use will focus on sustainable forms that are cleaner, more reliable and renewable. The most reliable and oldest form of energy is biomass—the product of photosynthetic organisms such as plants, some microbes and some algae. A closer examination of plant physiology and genetic engineering possibilities offers new pathways to improve bioenergy conversion.

The initial photosynthetic product of plants, glucose, is the immediate energy source for cells that use the sugar to metabolize other forms of cellular energy such as starch, oil and proteins. Some useful products we derive from biomass include biodiesel from algae, cooking oil from corn, and ethanol from sugarcane and corn. To be considered a good biomass candidate for product derivation, a species must satisfy three criteria: be high yielding, easy to grow and contain high levels of extractable content.

One example is sugarcane, a high-yielding tropical grass with a stem diameter of 1 to 3 inches and a height of up to 5 feet or more. Sugarcane’s high extractable sugar content accounts for 10 percent of its weight. It is no wonder that the sugarcane species Saccharum officinarum is one of the most important food-producing grasses in the world. Sugarcane requires strong sunlight, much water and a long maturation period.

Another example is hybrid yellow dent corn, a relatively easy spe­cies to grow and one of the most important production grains in the United States. Cultivated mostly in the temperate Midwest, corn is economical to grow and yields a large amount of biomass. The corn kernel is a seed with 3 to 4 percent oil, 9 to 10 percent protein, and about 72 percent starch. The high starch content makes corn a good product for livestock and for the extraction of high-fructose sugar. Currently, almost all of the 200-some ethanol producers in the U.S. use corn. To produce ethanol, however, corn starch must be broken down into fermentable sugars, which makes it less economical than sugarcane with its high extractable content of fermentable sugars.

Nature has endowed certain plants such as sugarcane with enhanced metabolic pathways to produce high sugar content. Molecular biologists can nudge certain unendowed plants, such as switchgrass, toward similar pathways. All plants produce sugar by photosynthesis, but differ in how the sugar is used or stored for easy access. Depending on the physiologic needs of a plant, sugar can be stored in starch, converted into oil, used for producing structural cellulose, or stay in circulation as an immediate energy source.

A major hurdle in ethanol production has always been low yield and the unavailability of quality feedstock. Efforts to increase yield often focus on strategies in producing feedstock with high sugar content, either by genetic engineering or breeding selection. An obvious choice for selective breeding is sugarcane. Its sugar content is good, but its growth rate and ease of cultivation need improvement. Switchgrass is a grass species with a fast growth rate that is easy to grow, but lacks high sugar content. An ideal solution, then, would be to cross sugarcane and switchgrass—sugarswitch—which may satisfy all the three criteria of high yield, easy to grow and high extractable sugar content.

The downside to selective breeding is that it involves a long learning curve, and the pattern of inheritance of desirable traits is not always straightforward. An alternative strategy is genetic engineering, which has become more widespread in the past three decades. In engineering plants producing high sugar content, the biology of the target species must be considered. For example, the yellow dent corn kernel is a seed—a fertilized embryo in dormancy until germination occurs and rapid growth requires the utilization of stored nutrients in the form of starch, protein and oil. In contrast, in rice seed, which has 16 to 20 percent lipid content in its aleurone layer, lipase activity utilizes oil as an energy source. The digested lipid content is used for gluconeogenesis within glyoxysomes. The rapidly growing, germinating seeds are able to convert stored oil into sugar for immediate energy, suggesting an approach to engineering enhanced sugar content in corn kernels or rice seeds.

Under certain conditions, plant oil such as corn triglycerides, can be reverted back into glucose sugar, the substrate for ethanol production. With the current ethanol production efficiency averaging about 93 percent of theoretical yield within the industry, there is not much room for improvement. Attempts at increasing yield should, therefore, focus on the metabolic conversion of oil into sugar.

Mechanistically, triglycerides are broken down into fatty acids and glycerol—precursors for gluconeogenesis, the pathway that converts biomolecules into glucose. Fatty acids are converted into oxaloacetate using enzymes that include isocitrate lyase and malate synthase (Figure 1). Oxaloacetate is then used for the gluconeogenic conversion into glucose (Figure 2). Another product of triglyceride breakdown, glycerol is metabolized into glyceraldehyde 3-phosphate (GA3-P), a more immediate substrate for glucose synthesis. The pathway is catalyzed by glycerol kinase, G3P dehydrogenase, and triosephosphate isomerase enzymes (Figure 3).

Various attempts at increasing ethanol yield have focused on enzymes or engineering new microbial strains for conversion. Enzyme use is cost-prohibitive, and complexity is an issue with microbial engineering. A different approach to plant engineering opens new possibilities for producing plants fit for high ethanol yield. Plants could be engineered to enhance the expression of key enzymes that metabolize oil into sugar. The key catalysts in the pathways are isocitrate lyase (Figure 1), PEPCK (Figure 2), and glycerol kinase (Figure 3). Studies have shown that isocitrate lyase knockout in Arabidopsis leads to a drastic reduction of gluconeogenesis from oxaloacetate. The lack of malate synthase, however, does not have any effect on gluconeogenesis. PEPCK reduction in Arabidopsis also reduced the sugar levels. In the storage tissues of fatty seedlings, glycerol kinase was shown as responsible for the initial conversion of glycerol into sugar. The studies indicate the power of engineering plants that convert their oil content into sugar.

The potential for yellow dent corn is intriguing, with its 3 to 4 percent oil content. If 95 percent of the oil could be converted to sugar, ethanol yield could be increased by as much as 10 percent, given that oil has more than twice the amount of energy as sugar.

Author: Trent Nguyen
President, Ebio Consulting
(972) 983-1969