To kill off invading microbes, companies must instead use either steam sterilization, which requires fermentation vessels to be built from expensive stainless steels, or costly antibiotics. Exposing large numbers of bacteria to these drugs also encourages the appearance of tolerant bacterial strains, which can contribute to the growing global problem of antibiotic resistance.

The researchers engineered E. coli with a synthetic six-step pathway that allows it to express enzymes needed to convert melamine—a xenobiotic (artificial) compound containing 67 % wt. nitrogen—to ammonia and carbon dioxide, in a strategy they have dubbed ROBUST (Robust Operation By Utilization of Substrate Technology).

The ROBUST strategy. Macronutrients essential for microbial growth are supplied in the form of xenobiotic or ecologically rare chemicals. Metabolic pathways enabling macronutrient assimilation are engineered in the desired biocatalyst (blue cells), establishing them as the dominant microorganism over nonutilizing contaminants (brown and red cells) inside the industrial bioreactor environment. NAD+, oxidized nicotinamide adenine dinucleotide; NADH; reduced nicotinamide adenine dinucleotide. Credit: AAAS, Shaw et al. Click to enlarge.

When they experimented with a mixed culture of the engineered E. coli strain and a naturally occurring strain, they found the engineered type rapidly outcompeted the control, when fed on melamine.

They then investigated engineering the yeast Saccharomyces cerevisiae to express a gene that allowed it to convert the nitrile-containing chemical cyanamide into urea, from which it could obtain nitrogen. The engineered strain was then able to grow with cyanamide as its only nitrogen source.

Finally, the researchers engineered both S. cerevisiae and the yeast Yarrowia lipolytica to use potassium phosphite as a source of phosphorous. Like the engineered E. coli strain, both the engineered yeasts were able to outcompete naturally occurring strains when fed on these chemicals.

Conventional biofermentation refineries typically use ammonium to supply microbes with a source of nitrogen. But contaminating organisms, such as Lactobacilli, can also extract nitrogen from ammonium, allowing them to grow and compete with the producer microorganisms. In contrast, these organisms do not have the genetic pathways needed to utilize melamine as a nitrogen source, said Gregory Stephanopoulos, the Willard Henry Dow Professor of Chemical Engineering and Biotechnology at MIT.

So by engineering the strains to make them capable of utilizing these unconventional sources of phosphorous and nitrogen, we give them an advantage that allows them to outcompete any other microbes that may invade the fermenter without sterilization.

—Gregory Stephanopoulos

The microbes were tested successfully on a variety of biomass feedstocks, including corn mash, cellulosic hydrolysate, and sugar cane, where they demonstrated no loss of productivity when compared to naturally occurring strains.

The paper provides a novel approach to allow companies to select for their productive microbes and select against contaminants, according to Jeff Lievense, a senior engineering fellow at the San Diego-based biotechnology company Genomatica who was not involved in the research.

In theory you could operate a fermentation plant with much less expensive equipment and lower associated operating costs. I would say you could cut the capital and capital-related costs [of fermentation] in half, and for very large-volume chemicals, that kind of saving is very significant.

—Jeff Lievense

The ROBUST strategy is now ready for industrial evaluation, said Joe Shaw, senior director of research and development at Novogy, who led the research. The technique was developed with Novogy researchers, who have tested the engineered strains at laboratory scale and trials with 1,000-liter fermentation vessels, and with Felix Lam of the MIT Whitehead Institute for Biomedical Research, who led the cellulosic hydrosylate testing.

Novogy now hopes to use the technology in its own advanced biofuel and biochemical production, and is also interested in licensing it for use by other manufacturers, Shaw says.

Resources

• A. Joe Shaw, Felix H. Lam, Maureen Hamilton, Andrew Consiglio, Kyle Macewen, Elena E. Brevnova, Emily Greenhagen, W. Greg Latouf, Colin R. South, Hans Van Dijken, Gregory Stephanopoulos (2016) “Metabolic engineering of microbial competitive advantage for industrial fermentation processes” Science doi: 10.1126/science.aaf6159