Asha Sri Sai Manikonda

Horizontal gene transfer (HGT) is one of the key biosafety challenges in the application of genetically engineered microorganisms (GEMs) for bioremediation, as it carries the potential risk of transferring artificially introduced genes to native microbiota or pathogenic organisms. To mitigate this risk, researchers are actively developing novel, multilayered strategies for molecular control of HGT, combining synthetic biology, biological containment systems, and mechanisms for programmed environmental restriction of microbial survival.

One approach involves the design of genetic safety systems, such as:

• Gene dependency systems, where GEMs can only survive in the presence of specific synthetic nutrients not found in nature,
• Kill-switch mechanisms that trigger cell death if the organism leaves a controlled environment or detects critical changes in external conditions,
• The incorporation of low-mobility genetic constructs, where engineered genes are integrated into the chromosome rather than plasmids, thereby reducing their potential for horizontal transfer.

Advanced bioencapsulation techniques (e.g., alginate matrices) are also used to physically isolate GEMs from native microbial communities while maintaining their functionality in contaminated environments.

Emerging research directions include the use of orthogonal genetic codes and synthetic nucleic acids (XNA), where engineered genes differ from natural sequences and remain functional only within a specifically designed system, further decreasing the likelihood of successful HGT.

From the perspective of environmental protection, it is essential that any GEM-based project be accompanied by comprehensive risk assessments, gene flow modeling, field validation, and closed-loop experimental protocols, ensuring full biological containment, reversibility, and accountability within natural ecosystems.

To enhance control of horizontal gene transfer (HGT) in genetically engineered microbes (GEMs) for bioremediation, researchers are developing novel, multi-layered strategies that combine

synthetic biology, genetic containment, and physical compartmentalization. These strategies aim to prevent engineered genes from spreading to native organisms or pathogens in the environment.

Genetic kill switches

Genetic kill switches trigger the self-destruction of GEMs if they escape a bioremediation site or are no longer needed.

• Engineered toxin-antitoxin systems: A microbe is engineered with a stable toxin and an unstable antitoxin, which is programmed to degrade outside the bioremediation site. This frees the toxin, which kills the cell.
• Multiple conditional safeguards: Robust and multilayered kill switches are designed to be activated by multiple different environmental inputs, such as temperature, specific chemical inducers, or light. The system uses AND-gate logic, requiring several specific signals to allow the microbe to survive. This reduces the risk of evolutionary escape, which occurs when a single kill switch is disabled by a random mutation.
• Auxotrophic containment: The microbe is engineered to depend on a synthetic nutrient not found in nature. This forces the microbe to die when it leaves the controlled environment where the nutrient is supplied. Some advanced systems use non-standard amino acids (NSAAs) to prevent natural products in the environment from sustaining the microbes.

Advanced genetic controls

Next-generation tools like CRISPR-Cas systems and synthetic biology are being used to more precisely regulate gene flow.

• CRISPR-Cas9-assisted biocontainment: This system can be programmed to degrade the engineered genes if they are horizontally transferred. In some designs, a CRISPR device can also kill any recipient cell that acquires the engineered genes, blocking dissemination to wild-type bacteria.
• Targeting essential host genes: For bioremediation that uses indigenous bacteria, an approach known as "genetic bioaugmentation" uses a self-destructing plasmid to prime the native microbial community. A gene-carrying vector can transfer bioremediation capabilities to indigenous species, but the vector is designed to only persist as long as a selection pressure (the pollutant) is present, disappearing once the contaminant is degraded.
• Genome integration vs. plasmid systems: Engineered genes are integrated directly into the microbe's chromosome instead of being carried on plasmids, which can be easily shared via HGT. This anchors the genetic payload and prevents its independent spread.

Physical and ecological barriers

Physical confinement and harnessing ecological principles provide additional layers of protection.

• Encapsulation: GEMs can be immobilized or encapsulated in protective matrices, such as hydrogels or alginate beads. This allows them to perform their function in a contaminated environment while being physically separated from other microorganisms.
• Harnessing microbial community dynamics: Instead of releasing GEMs directly, researchers can use HGT to "jump-start" indigenous microbial populations. Synthetic vectors with bioremediation genes are introduced, but the vector is designed to only persist if the pollutant is present.
• Quorum sensing safeguards: Since bioremediation often requires a high density of engineered microbes, quorum sensing systems can be engineered to control GEM behavior based on population density. For instance, a kill switch could be triggered if a GEM leaves the quorum and can no longer sense a high density of its engineered counterparts.