Biocontainment of Genetically Engineered Algae, an Update

by Jacob Sebesta, Wei Xiong, Michael T. Guarieri and Jianping Yu, Biosciences Center, National Renewable Energy Laboratory, Golden, CO

At industrial scale, genetically engineered algae may be cultivated outdoors in open ponds or in closed photobioreactors. In either case, industry would need to address a potential risk of the release of the engineered algae into the natural environment, resulting in potential negative impacts to the environment. Genetic biocontainment strategies are therefore under development to reduce the probability that these engineered organisms can survive outside of the laboratory or industrial setting.

These strategies intend to kill the escaped cells by expression of toxic proteins, and passive strategies that use knockouts of native genes to reduce fitness outside of the controlled environment of labs and industrial cultivation systems.

Several biocontainment strategies have demonstrated escape frequencies below detection limits. However, they have typically done so in carefully controlled experiments which may fail to capture mechanisms of escape that may arise in the more complex natural environment. The selection of biocontainment strategies that can effectively kill cells outside the lab, while maintaining maximum productivity inside the lab and without the need for relatively expensive chemicals needs further study.

Introduction to this research

Genetic modification of algae, including eukaryotic microalgae and cyanobacteria, is expected to facilitate direct conversion of light energy and inorganic carbon to a wide variety of valuable chemicals. As with other genetically modified organisms (GMOs), the environmental risk of large-scale cultivation must be assessed, and appropriate measures must be taken to mitigate those risks. Previously, Henley et al. (2013) reported a risk assessment for genetically engineered microalgae, finding that risks to human health, the environment, and the economy, were generally low, but not zero.

Given that genetically engineered algae may be grown outdoors, possibly in open ponds, they determined that the potential for these cells to escape into the environment is elevated beyond that of typical industrial microbial cultivation. Therefore, they recommended the development of biocontainment strategies which reduce growth fitness in the natural environment, that are conditionally lethal to the cells when they are not in the lab or industrial setting and that have reduced capability to transfer genetic material to other organisms.

Since that report, many new genetic biocontainment strategies have been developed for microalgae and other industrially relevant microorganisms which achieve one or more of those goals.

In synthetic auxotrophy, cells are modified to make their growth dependent on an unusual or nonnatural nutrient or an unnaturally high concentration of a nutrient. Examples include dependence on unusual phosphorous sources like phosphite and dependence on high concentrations of carbon dioxide.

Further efforts have been made to express toxic proteins, such as nucleases, in the cells in a manner dependent on the conditions outside the lab, typically, the loss of some synthetic signal molecule or an unnatural concentration of a signal molecule. Biocontainment strategies have been collected in the Biocontainment Finder on the Standardsinsynbio.eu website.

The full version of this review summarizes the rationale for designing genetically encoded biocontainment systems and the efforts made thus far to assess their efficacy in genetically engineered algae. First, the report discusses the possible escape routes and fates of escaped algae. The regulatory requirements for outdoor cultivation of genetically engineered algae in a few regions are then summarized. Next, an overview of the different types of genetically encoded biocontainment strategies that may be used in algae is provided.

An examination of whether lab tests, which frequently demonstrate the achievement of meeting the NIH guideline of a 10⁻⁸ cell survival rate, are truly representative of what may occur if cultures were released into the natural environment. And finally, there is a discussion on the results of some specific examples of genetically encoded biocontainment found in recent publications and some suggestions for future directions.