Devarenne’s lab has been studying the concept of making fuel from algae on a $2-million National Science Foundation grant for four years.

Microalgae are a promising next-generation source of feedstocks for biofuel production with the potential to serve as a practical alternative to petroleum-based transportation fuels. Depending on the microalgal species, the oils produced vary from triacylglycerols to hydrocarbons. Hydrocarbon-based fuels are preferred over other biofuels, as they are highly compatible with existing petroleum infrastructures and possess superior fuel properties.

The colony-forming green microalga B. braunii is an exciting candidate for biofuel feedstock production, as it produces up to 61% of its dry weight as liquid hydrocarbon oils. These hydrocarbons are produced inside the cells of the colony, seen as intracellular oil bodies and secreted into the colony extracellular matrix where the majority of the hydrocarbons are stored. Most importantly, catalytic hydrocracking of hydrocarbons from this alga results in petroleum-equivalent fuels of gasoline, kerosene and diesel. Intriguingly, geologic evidence also shows a direct contribution of this alga to the formation of currently used fossil fuel deposits around the globe.

Despite the aforementioned advantages of B. braunii, its use for biofuel feedstock production is hindered by a slow growth rate and the lack of transformation systems to achieve targeted genetic modification. Thus, the identification of B. braunii hydrocarbon biosynthetic pathways and associated genes/enzymes can provide options for metabolically engineering these pathways into heterologous hosts with better growth characteristics and the ability to be genetically manipulated. This would then allow the development of improved versions of hydrocarbon biosynthetic enzymes, to direct production towards the most commercially desirable products.

—Thapa et al.

Devarenne’s lab has been trying to understand how Botryococcus braunii makes the liquid hydrocarbons—i.e., what genes and pathways are involved—so the genes can be manipulated to make more oil, possibly by transferring those genes into a land plant such as tobacco, or maybe other algae that grow very quickly.

It takes about a week for one Botryococcus cell to double into two cells, whereas a faster growing algae—but one that doesn't make a lot of oil—can double in about six hours, Devarenne said.

The researchers targeted deciphering the biochemical pathway for making the hydrocarbon oil, which is called lycopadiene. They discovered a gene called lycopaoctaene synthase (LOS). The enzyme encoded by the LOS gene is able to initiate the production of the oil.

A closer look at the LOS enzyme revealed that the enzyme is “promiscuous” in that it is capable of mixing several substrates to make different products.

Some of the substrates are 20 carbons long, some are 15 carbons long. We can mix them with the enzyme so that two 20-carbon molecules will make a 40 carbon molecule, or two 15-carbon molecules to make a 30 carbon molecule, or a 20-carbon substrate and a 15-carbon substrate will make a 35-carbon substrate.

—Tim Devarenne

Devarenne explained that’s not only different from other enzymes that are similar to LOS, but it’s important because most enzymes like LOS only use a 15-carbon substrate. In terms of fuel, it's better to start with a higher carbon number molecule.

The team determined the sequence of all the actively working genes of the organism under hydrocarbon producing conditions. Bioinformatic analysis of this sequence information was then able to pinpoint a gene that might have the appropriate activity to initiate hydrocarbon biosynthesis.

We’re still a ways away from making a commercial product, but our next step is to finish deciphering the pathway. We’ve identified the very first step in the pathway—making the first 40 carbon hydrocarbon. We have some gene candidates for the next step of the pathway, and we are just starting to characterize those.

—Tim Devarenne

Even when the genes are more fully understood, scientists will have to find the right host organism to express the genes, optimize that expression and try to get them to produce as much of the oil as possible.

The project included Devarenne’s graduate student Hem Thapa and colleague Mandar Naik at Texas A&M University in College Station, along with Shigeru Okada and Kentaro Takada from the University of Tokyo in Japan, Istvan Molnar from the University of Arizona’s College of Agriculture and Life Sciences and Yuquan Xu from the Chinese Academy of Agricultural Sciences.

Resources

• Hem R. Thapa, Mandar T. Naik, Shigeru Okada, Kentaro Takada, István Molnár, Yuquan Xu & Timothy P. Devarenne (2016) “A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L” Nature Communications 7, Article number: 11198 doi: 10.1038/ncomms11198