Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA).

Transcriptional Regulation Contributes to Prioritized Detoxification of Hydrogen Peroxide over Nitric Oxide.

Hydrogen peroxide (HO) and nitric oxide (NO·) are toxic metabolites that immune cells use to attack pathogens. These antimicrobials can be present at the same time in phagosomes, and it remains unclear how bacteria deal with these insults when simultaneously present. Here, using , we observed that simultaneous exposure to HO and NO· leads to prioritized detoxification, where enzymatic removal of NO· is impeded until HO has been eliminated.

An integrated network analysis identifies how ArcAB enables metabolic oscillations in the nitric oxide detoxification network of Escherichia coli.

The virulences of many pathogens depend on their abilities to detoxify the immune antimicrobial nitric oxide (NO•). The functions of bacterial NO• detoxification machinery depend on oxygen (O ), with O inhibiting some enzymes, whereas others use it as a substrate. Previously, Escherichia coli NO• detoxification was found to be highly attenuated under microaerobic conditions and metabolic oscillations were observed.

A Kinetic Platform to Determine the Fate of Hydrogen Peroxide in Escherichia coli.

Hydrogen peroxide (H2O2) is used by phagocytic cells of the innate immune response to kill engulfed bacteria. H2O2 diffuses freely into bacteria, where it can wreak havoc on sensitive biomolecules if it is not rapidly detoxified. Accordingly, bacteria have evolved numerous systems to defend themselves against H2O2, and the importance of these systems to pathogenesis has been substantiated by the many bacteria that require them to establish or sustain infections.

Futile cycling increases sensitivity toward oxidative stress in Escherichia coli.

Reactive oxygen species (ROS) are toxic molecules utilized by the immune system to combat invading pathogens. Recent evidence suggests that inefficiencies in ATP production or usage can lead to increased endogenous ROS production and sensitivity to oxidative stress in bacteria. With this as inspiration, and knowledge that ATP is required for a number of DNA repair mechanisms, we hypothesized that futile cycling would be an effective way to increase sensitivity to oxidative stress.

Deciphering nitric oxide stress in bacteria with quantitative modeling.

Many pathogens depend on nitric oxide (NO•) detoxification and repair to establish an infection, and inhibitors of these systems are under investigation as next-generation antibiotics. Because of the broad reactivity of NO• and its derivatives with biomolecules, a deep understanding of how pathogens sense and respond to NO•, as an integrated system, has been elusive. Quantitative kinetic modeling has been proposed as a method to enhance analysis and understanding of NO• stress at the systems-level.

Antimicrobial sand via adsorption of cationic Moringa oleifera protein.

Moringa oleifera (Moringa) seeds contain a natural cationic protein (MOCP) that can be used as an antimicrobial flocculant for water clarification. Currently, the main barrier to using Moringa seeds for producing potable water is that the seeds release other water-soluble proteins and organic matter, which increase the concentration of dissolved organic matter (DOM) in the water. The presence of this DOM supports the regrowth of pathogens in treated water, preventing its storage and later use.