May 15, 2008 (Vol. 28, No. 10)

Products from Lucigen and C5-6 Technologies Targeted at Molecular Biology and Biofuels

While working at the University of Wisconsin, Madison, David Mead, Ph.D., helped to develop the technology behind TA cloning, now one of the most popular methods for cloning-amplified PCR products using Taq and other polymerases. That technology was sold to Invitrogen (, where it was incorporated into products that earn millions of dollars each year.

In 1998, Dr. Mead and Tom Schoenfeld, Ph.D., cofounded Lucigen ( “to duplicate that kind of success,” Dr. Mead says. The initial idea behind Lucigen was to find better polymerases in extreme environments like those of Yellowstone National Park, according to Dr. Mead, president of Lucigen. When Phil Brumm, Ph.D., an expert in hydrolases, joined Lucigen, the company added biomass and biofuels projects to its agenda.

In August 2006, Lucigen consolidated the biomass and biofuels work into C5-6 Technologies (, a company that specializes in making enzymes that break down complex biomass into 5- and 6-carbon sugars. Dr. Brumm and John Biondi, then COO at Lucigen, spun out C5-6 Technologies, where Dr. Brumm serves as CSO and Biondi as president. C5-6 Technologies and Lucigen operate independently but share facilities in Middleton, WI.

Biomass Busters

C5-6 Technologies plans to release its first products—a suite of enzymes to improve the efficiency of current methods for fermenting corn to ethanol—in the second half of 2008. Although the long-term goal is to make ethanol from nonfood, cellulosic biomass, “corn will stay around and its use will grow,” says Biondi. It is predicted that the current 7 billion gallon annual production rate of ethanol from corn will grow to 15 billion gallons by 2015.

The company’s first product, named CornBuster™, will improve the yield of ethanol from corn by 3%. At a typical drymill corn-to-ethanol manufacturing facility that makes 50,000-gallon batches, CornBuster will boost the annual production from 1 million to 1.5 million gallons while starting with the same amount of corn, according to Biondi. C5-6 Technologies is conducting the final pilot-plant trials of CornBuster and completing the regulatory process, as well as raising A-round venture capital.

Also in the pipeline is SoyBuster™, which contains thermostable enzymes that convert low-value carbohydrates in soy meal into ethanol, while concentrating soy’s protein, increasing its value. The enzymes discovered by the company will replace expensive and harsh chemical methods presently employed to disrupt recalcitrant cellulose in biomass.

Novel ideas for degrading biomass are exploding, and it is not clear which technologies will win out. It is clear that “the current paradigm of using fungal enzymes is not cutting it in the real world,” says Dr. Mead. Such enzymatic approaches are not economically feasible because they take too long to work. “The company that finds blends of enzymes with higher specific activities to process biomass rapidly and cheaply will win out,” he says.

Lucigen’s Offerings

As C5-6 Technologies advances its biomass platform, Lucigen continues to expand its molecular biology products for gene cloning and genomics. Since the introduction of its first product, CloneSmart®, Lucigen has released other cloning products, including ClonePlex™-AK dual insert cloning kits, CopyRight™ large insert cloning kits, and BigEasy® linear cloning system.

Recently Lucigen discovered methods to clone genes and entire genomes directly from a few or just a single isolated cell. This capability, called PicoClone™, represents a billion-fold improvement in cloning compared to standard methods, says Dr. Mead. PicoClone makes it possible to clone genes from microbes that cannot be grown in the laboratory, such as extremophiles living in boiling hot springs. Lucigen researchers use PicoClone to discover superior types of enzymes for basic and biomedical research, molecular diagnostics, drug discovery, and industrial processes.

The first product developed using PicoClone is Pyrophage 3173 DNA Polymerase™, the first replicase cloned from a bacteriophage living in a boiling hot spring in Yellowstone National Park. Because PyroPhage 3173 contains a true DNA replicase, rather than the DNA polymerases found in all other current PCR enzymes, it gives more robust and truer replications, notes Dr. Mead. The thermostable properties of Pyrophage 3173 DNA make it ideal for thermocycling methods like PCR, he adds.

At the end of 2007, Lucigen released ExCyto PCR™, a new concept in DNA cloning and PCR analysis. ExCyto PCR incorporates a thermostable DNA polymerase into the E. coli chromosome, so there is no need for external DNA polymerases. “It’s a clever new way to bypass the work of purifying a gene and buying expensive DNA polymerases,” says Dr. Mead. ExCyto PCR allows researchers to insert a gene, incubate overnight, select a colony, and add cells to provided primers.

The ExCyto PCR kit comes with competent cells and all reagents for high efficiency ligation, transformation, and PCR. Highly reliable and reproducible amplification inserts of up to 5 kb are directly obtained from a colony sample. The PCR reagents provided are sufficient to perform at least 100 50 µL amplifications.

In June 2007, Lucigen and C5-6 Technologies were named commercial partners in the DOE’s Great Lakes Bioenergy Research Center (GLBRC). Based at the University of Wisconsin and funded with a five-year, $125 million grant, GLBRC includes investigators at Michigan State University, University of Florida, and Oak Ridge National Laboratory who are searching for ways to generate biofuels from biomass. By applying their enzyme technologies, Lucigen and C5-6 Technologies will contribute to expanding commercial opportunities for cellulosic biomass.

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