Team:William and Mary/Design

Overview Circuit Design for TheraPUFA Module 1: High Pass Cytokine Sensor Module 2: PUFA Production Module 3: PUFA Export Module 4: Kill Switches Future Directions and Alternative Circuits


Design

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Overview


TheraPUFA is a smart nasal probiotic designed to suppress infection by enveloped and positive-strand RNA viruses while regulating inflammation. Alongside social distancing, masks, and rigorous testing, TheraPUFA could potentially slow the spread of a viral pandemic if administered prophylactically. Unlike existing antiviral drugs, TheraPUFA intelligently and dynamically responds to inflammation within the human body, suppressing excessive inflammation while reducing risk of immunosuppression. Scroll down to read about the inner workings of TheraPUFA.

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Circuit Design for TheraPUFA


TheraPUFA is composed of Neisseria cinerea, a commensal, biosafety level 1 (BSL1), and gram negative bacterium native to the nasopharyngeal microbiome (Custodio et al., Plos Pathogens, 2020). We selected N. cinerea due to its compatibility with the cytokine sensor we selected, which must be expressed within gram negative bacteria. Additionally, we selected N. cinerea since it exists within the natural nasal flora of healthy individuals without causing disease. Future research, described at the bottom of this page, could include the adaptation of TheraPUFA to gram positive chasses such as Lactobacillus, which have more established usage in nasal probiotics than gram negative strains. To read more about chassis selection, including a literature review on the nasopharyngeal microbiome, please visit our Engineering Success page. Feedback on chassis selection provided by our contacts in the medical community are described in detail on our Human Practices page. Finally, safety considerations related to chassis selection are described on our Safety page.

Though all bacteria within TheraPUFA belong to N. cinerea, they are divided between two distinct groups that differ in the genetic circuits that they contain. The two groups are shown in the graphic above as “Bacterium 1” and “Bacterium 2.” “Bacterium 1” is responsible for the production of arachidonic acid (AA), while “Bacterium 2” is responsible for the production of docosahexaenoic acid (DHA). TheraPUFA is “smart” in that it utilizes a cytokine sensor to respond to inflammation in the body, controlling when Bacterium 1 and 2 produce their respective PUFA. Both Bacterium 1 and 2 contain two different kill switches to address safety concerns. Though both groups contain both two kill switches, the first kill switch is depicted within Bacteria 1 and the second is depicted within graphic 2 in the graphic above.

Below, we describe how TheraPUFA works in detail, including its dynamic response to inflammation.

Condition 1: Low Cytokine Concentrations - Cytokine Concentrations Insufficient to Trigger High Pass Filter

  1. Concentrations of pro-inflammatory cytokines IFN-γ and TNF-α are beneath the detection threshold of a cytokine sensor expressed by both Bacterium 1 and 2. The low sensitivity of this sensor allows it to act as a high pass filter, responding only once cytokine concentrations have surpassed a threshold.

  2. Bacterium 1 constitutively produces arachidonic acid (AA) utilizing an Anderson series promoter J23. AA, a polyunsaturated fatty acid (PUFA) with both pro- and anti-inflammatory metabolites, lyses viral envelopes and suppresses replication by positive-strand RNA viruses. AA accumulates within membrane phospholipids.

  3. Bacterium 1 secretes AA constitutively at basal levels. In the absence of the cI repressor, the uninhibited pLac promoter promotes the expression of phospholipase cPLA2, which releases AA from membrane phospholipids. AA diffuses into the extracellular environment, where it can provide a therapeutic effect. Since AA contributes primarily to pro-inflammatory metabolites, secreted AA allows the immune system to mount a response to viral infection, unlike certain anti-inflammatory agents.

  4. Bacterium 2 produces docosahexaenoic acid (DHA) utilizing a constitutive Anderson series promoter. DHA accumulates within membrane phospholipids but is not secreted in the absence of iPLA2 phospholipase. iPLA2 is not produced when the high pass filter is not triggered.

Condition 2: High Cytokine Concentrations - Cytokine Concentrations Surpass Threshold of High Pass Filter

  1. In the case of extreme inflammation, high concentrations of pro-inflammatory cytokines IFN-γ and TNF-α surpass the detection threshold of the cytokine sensor contained by both Bacterium 1 and 2. The low sensitivity of this sensor allows it to act as a high pass filter, responding once cytokine concentrations have surpassed the threshold.

  2. The cytokine sensor activates the pspA promoter in Bacterium 1, which promotes the expression of repressor cI. cI represses the pLac promoter, halting the secretion of AA. The secretion of AA, which has primarily pro-inflammatory cytokines, is turned “off.”

  3. Triggered by the high cytokine concentrations, the same cytokine sensor activates the pspA promoter in Bacterium 2. pspA promotes the expression of phospholipase iPLA2, which releases DHA from membrane phospholipids. The secretion of DHA is turned “on.” Exported DHA provides an antiviral effect while suppressing extreme inflammation. Should pro-inflammatory cytokine concentrations fall back beneath the high pass filter’s threshold, the pspA promoter will not be activated in either bacterial type. No longer inhibited by cI, Bacterium 1 will return to secreting AA. Meanwhile, DHA secretion will halt in Bacterium 2, due to the lack of activation of the pspA promoter. The switch back to AA from DHA prevents the immunosuppression some patients experience following extreme inflammation.

The circuit diagram above displays but one of the PUFA export systems we have designed. Other circuits vary in their export systems, though all rely on the high pass filter to trigger secretion of anti-inflammatory DHA.

Why “switch” between AA and DHA?

SARS-CoV-2 causes a wide range of symptoms in patients that can change dramatically over the course of infection, necessitating the use of a smart drug such as TheraPUFA. For example, certain individuals remain completely asymptomatic while infected, whereas other individuals experience mild to severe inflammation. Extreme changes can be observed within a single patient, who may appear asymptomatic during a 5-6 day incubation period, then experience severe inflammation in the form of a cytokine form, followed by severe immunosuppression. An ideal drug needs to “sense” the level of cytokines and respond accordingly.

TheraPUFA is designed to respond intelligently and dynamically to varying degrees of inflammation and stages of infection. In the case of mild to no inflammation, such as the case of an asymptomatic patient, the probiotic will produce AA without ever switching to DHA. The anti-inflammatory properties of DHA are unnecessary for mild cases, and may cause more harm than good by dampening the immune response.

However, a patient experiencing a cytokine storm would benefit from an anti-inflammatory agent. This patient would produce sufficient quantities of pro-inflammatory cytokines TNF-α and IFN-γ to trigger the high pass filter, resulting in the release of anti-inflammatory DHA.

To provide a therapeutic effect, TheraPUFA relies on three modules: a cytokine-sensing system, a PUFA production system, and a PUFA export system. The individual modules of TheraPUFA are described below.

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Module 1: High Pass Cytokine Sensor


We designed TheraPUFA to intelligently and dynamically respond to inflammation within the human body. Standard pharmaceuticals that lack this capability fail to treat patients at all stages of infection and degrees of inflammation and may cause more harm than good. For example, the steroid Dexamethasone may decrease mortality rates for patients with severe inflammation who require ventilators or oxygen, but not for patients who do not require these interventions. Administered too early, anti-inflammatory agents may dampen the immune response to viral infection. Pulmonologist and inflammation expert Dr. Shelhamer warned us of this effect, causing us to carefully consider the usage of anti-inflammatory PUFAs such as DHA. Eventually, we designed TheraPUFA to constitutively produce AA, and switch to DHA only as-needed in the case of extreme inflammation. Since AA has both anti- and pro-inflammatory metabolites, and results in primarily pro-inflammatory metabolites at the start of infection, AA would allow a patient to mount a sufficient immune response without risk of immunosuppression.

References used for creation of image: Manganelli and Gennaro, 2017; Aurand and March, 2015

Pathogenic bacteria have evolved the ability to sense human cytokines as a form of protection against the immune response (Paino, 2012; Zav'yalov, 1995; Luo; 1993; Wu, 2005; Laughlin, 2000). For example, Pseudomonas aeruginosa contains an outer membrane porin called OprF that was found to sense interferon-γ (IFN-γ) (Wu, 2005) and activates the production of virulence factors such as type-I lectin (Laughlin, 2000).

To trigger the switch from AA to DHA, we have utilized a synthetic cytokine sensor designed by researchers Aurand and March (Aurand and March, 2015). This sensor replaces two extracellular loops from the bacterial outer membrane porin OmpA (native to E. coli) with two extracellular loops from OprF (native to Pseudomonas aeruginosa). The sensor is able to sense cytokines TNF-α and IFN-γ at specific concentrations (150 pM for TNF-α and 200 pM for IFN-γ) (Aurand and March, 2015). At levels below these concentrations in which the sensor is not activated, the C-terminal domains of inner membrane proteins PspB and C are interacting, and protein PspA interacts with transcription factor PspF, inhibiting it (Manganelli and Gennaro, 2017). Upon interacting with either IFN-γ or TNF-α at these specific concentrations, the sensor is activated, and the stress-induced upon the outer membrane porin is transduced to inner membrane proteins PspB and PspC (Manganelli and Gennaro, 2017). This forces PspB and C to stop interacting with each other, allowing PspA to interact with the C-terminal domain of PspC, preventing it from inhibiting PspF (Manganelli and Gennaro, 2017). Therefore, PspF is able to interact with the PspA promoter and induce gene expression of the DHA PUFA synthase (Manganelli and Gennaro, 2017). Within the bacterial type responsible for AA production, PspF interacts with the PspA promoter to trigger the expression of a repressive protein that represses the expression of the AA PUFA synthase. Thus, AA production is switched off and DHA production is switched on. The high threshold of cytokines required to activate the sensor allows the sensor to act as a high-pass filter, switching between PUFAs only in the case of extreme inflammation.

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Module 2: PUFA Production


Organisms produce PUFAs through de novo synthesis or through the elongation and desaturation of shorter, saturated fatty acids. We have identified an Aurespira marina and a Schizochytrium PUFA synthase for the de novo synthesis of AA and DHA, respectively.

  • Aurespira marina is a marine bacterium native to the coast of Thailand (Hosoya, Int. J. Syst. Evol. Microbiol., 2006; Ujihara et al., FEBS Letters, 2014).

  • Schizochytrium is a Thraustochytrid protist known for DHA production (Yoshida et al., Marine Drugs, 2016; Hauvermale et al., Lipids, 2006; Metz et al., Plant Physiol. Biochem., 2009).

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Module 3: PUFA Export


Engineered systems for PUFA export following synthesis within a bacterial cell have not been characterized within the literature. To develop a system for the export of AA and DHA, we investigated systems for similar fatty acids.

Tong et al. implemented a system for long chain fatty acids, including the mono-unsaturated fatty acid cis-vaccenic acid, in gram negative bacterium Rhodobacter sphaeroides (BMC, 2019). Their system utilizes a phospholipase localized to the periplasm to release fatty acids from phospholipids of the inner membrane. The free fatty acids then exit the periplasm through the outer membrane via an unknown Tol-C independent method.

We adapted this system to our project by:
  1. Selecting an acyl-CoA synthetase with preference for PUFA such that PUFA produced by synthases are activated and directed towards phospholipid synthesis (Tonon et al., Plant Physiol., 2005)
  2. Selecting a phospholipase with preference for PUFA such that PUFAs are released from phospholipids into the periplasm (Rosa & Rapoport, BBA, 2009)

For the export of AA:

  1. AA is synthesized by PUFA synthase and incorporated into phospholipids of the inner cell membrane. Unlike DHA, which appears to initially accumulate as intracellular free fatty acid, AA appears to accumulate within phospholipids--no additional engineering is required to divert synthesized AA to phospholipid synthesis (Metz et al., Plant Physiol. Biochem., 2009, Ujihara et al., FEBS Letters, 2014). Therefore, when modeling AA production, we collapse synthesis and phospholipid incorporation into one step.

  2. cPLA2 localized to the periplasm utilizing a CycA localization tag from Neisseria sp. releases free AA from the inner membrane (Turner et al., The Biochemical Journal, 2005; Rosa & Rapoport, BBA, 2009).

  3. Exit from the periplasm through the outer membrane through a Tol-C independent mechanism (Tong et al., BMC, 2019). It is unlikely for fatty acids to passively diffuse through the outer membrane, especially considering the impermeability of bacterial membranes to long chain fatty acids (Schwenk et al., Prostaglandins Leukot. Essent. Fatty Acids, 2010; Kamp & Hamilton, Prostaglandins Leukot. Essent. Fatty Acids, 2006). So, we will assume a protein-mediated diffusion mechanism rather than simple diffusion. Specifically, we will assume that a FadL-like protein allows periplasmic free AA through the outer membrane and into the extracellular environment. FadL is a long-chain fatty acid transporter that moves free fatty acid into the periplasm (van den Berg et al., Science, 2004). We will assume that there is a similar protein that moves free fatty acid in the opposite direction.

For the export of DHA:

  1. DHA is synthesized as a free fatty acid by Schizochytrium PUFA synthase (Metz et al., Plant Physiol. Biochem., 2009). An additional phosphopantetheinyl transferase from Nostoc cyanobacteria is necessary for DHA synthesis when the DHA synthase is expressed heterologously.

  2. Free DHA is activated into DHA-CoA by acyl-CoA synthetase lacsA from diatom Thalassiosira pseudonana (Tonon et al., Plant Physiol., 2005).

  3. Activated DHA is incorporated into membrane phospholipids and inserted into the membrane by enzymes (PlsC incorporates fatty acids into the sn-2 position. We will only consider PlsC for this step, because potential chassis Neisseria cinerea utilizes the PlsX/Y/C system and lacks a PlsB enzyme capable of using DHA-CoA) (Sohlenkamp & Geiger, FEMS Microbiology Reviews, 2016).

  4. DHA is released from the inner membrane and into the periplasm by a phospholipase. Assume iPLA2, a human phospholipase with a preference for DHA, has been localized to the periplasm using a CycA’ localization tag endogenous to Neisseria sp. (Rosa & Rapoport, BBA, 2009; Turner et al., The Biochemical Journal, 2005).

  5. Exit from the periplasm through the outer membrane through a Tol-C independent mechanism (Tong et al., BMC, 2019). It is unlikely for fatty acids to passively diffuse through the outer membrane, especially considering the impermeability of bacterial membranes to long chain fatty acids (Schwenk et al., Prostaglandins Leukot. Essent. Fatty Acids, 2010; Kamp & Hamilton, Prostaglandins Leukot. Essent. Fatty Acids, 2006). So, we will assume a protein-mediated diffusion mechanism rather than simple diffusion. Specifically, we will assume that a FadL-like protein allows periplasmic free DHA through the outer membrane and into the extracellular environment. FadL is a long-chain fatty acid transporter that moves free fatty acid into the periplasm (van den Berg et al., Science, 2004). We will assume that there is a similar protein that moves free fatty acid in the opposite direction.

Design considerations for both AA and DHA export:

  • Knockout of the FadE gene prevents PUFA degradation (He et al., Biotechnol. Bioeng., 2014)

  • Knockout of FadD ensures that free PUFA released into the periplasm does not reenter the cytoplasm. FadD is an inner-membrane associated protein that uses ATP to move protonated free fatty acids from the periplasm into the cytoplasm (Weimar et al., J. Biol. Chem., 2002).

  • Knockout of the FadL gene prevents secreted PUFA from reentering the periplasm from the extracellular environment. FadL is a protein that can import long chain fatty acids into the periplasm (van den Berg et al., Science, 2004).

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Module 4: Kill Switches


The fourth module of TheraPUFA contains two kill switches. The first kill switch would allow for the eradication of TheraPUFA within a patient’s body, should the patient wish to clear the probiotic for any reason. The second kill switch would prevent TheraPUFA from surviving in the environment, outside of a patient’s body. The need for this kill switch was emphasized by Dr. Shelhamer, who explained that the nasal probiotic would easily escape into the environment shortly after administration.

  1. The in-body kill switch utilizes a toxin under the control of aspirin-inducible promoter PsaI to make aspirin fatal to TheraPUFA (Chen et al., 2019). A patient could easily clear the probiotic by simply self-administering aspirin.

  2. The second kill switch prevents survival of TheraPUFA outside the human body preventing the bacteria from replicating in temperatures below body temperature (37C). To accomplish this, the kill switch utilizes the FitAB toxin-antitoxin system (Mattison et al., 2006), combined with the RNA thermometer CssA (Barnwal et al., 2016).

For detailed information about our kill switches, as well as other safety considerations, please visit our Safety page.

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Future Directions and Alternative Circuits


We have developed additional export systems in order to investigate the potential benefits of utilizing a gram negative versus positive chassis, and to select the export system with the greatest export efficiency.

In our final model, we utilized the gram negative phospholipase-based system adapted from Tong et al., since our cytokine sensor is designed for gram negative bacteria. However, future research could identify cytokine-sensing systems in gram positive bacteria (S. aureus may respond to IL-8 and IFN-γ, for example), such that we could implement our probiotic in a gram positive chassis and utilize a gram positive export systems (Di Domenico et al, Scientific Reports, 2019).

Gram positive bacteria lack an outer membrane, meaning that fatty acids released from the cell membrane by a phospholipase would not face an additional membrane barrier while exiting the cell. Instead, the free fatty acids would simply diffuse through the porous cell wall into the extracellular environment. In addition to optimizing export, utilizing gram positive bacteria in our probiotic could address safety concerns related to gram negative strains. BSL1 gram negative species exist within the cavity, but the relationship of these species to disease led Dr. Turner of UVA Medical center to advise us against using them. Furthermore, gram negative species have not commonly been utilized as nasal probiotics, whereas gram positive Lactobacillus species have (De Boeck et al., 2020). To break from this precedent and utilize a gram negative species within a nasal probiotic, the medical doctors we interviewed stated that we would need rigorous, “waterproof” safety data.

An overview of the PUFA synthesis and export systems we have designed is shown above. The PUFA synthase utilized determines the PUFA produced, AA or DHA. To export AA and DHA, we designed phospholipase-based export systems for use in gram positive in addition to gram negative bacteria. We also developed a system that utilizes an S. aureus efflux pump to export AA from gram positive cells.

Phospholipase-Based PUFA Export System for Gram Positive Bacteria

To adapt the phospholipase-based PUFA export system inspired by Tong et al. to a gram positive chassis, we:

  1. Selected a fatty acid kinase with a preference for PUFA to generate PUFA-PO4 from synthesized PUFA (Gullet et al., J. Biol. Chem., 2019). PUFA-PO4 may be utilized by enzymes within gram positive cells to produce phospholipids. Though this step is analogous to the first step in the gram positive system, we use an acyl-PO4 intermediate rather than an acyl-CoA intermediate to direct fatty acids towards phospholipid synthesis, due to the differences in this process between gram negative and gram positive bacteria.

  2. Determined strategies to localize phospholipase molecules to the gram positive periplasm. In the absence of an outer membrane, we must rely on the charge and size of phospholipase molecules to prevent their exit through the cell wall. Attracted to the negative charge of the cell wall, positively-charged molecules are less likely to exit into the extracellular environment (van Wely et al., FEMS Microbiology Reviews, 2001; Stephenson, Biochem. J, 2000). Additionally, though the cell wall is porous, it is impermeable to large protein molecules (Desvaux, Candella, Serror, Front. Microbiol, 2018; Forster & Marquis, Molecular Microbiology, 2012).

  3. Selected phospholipases capable of cleaving at the sn-1 position of phospholipids and at the sn-2 position of phospholipids (Ishibashi et al., Scientific Reports, 2019). The position of fatty acid within phospholipids depends on whether PlsB or PlsY inserts PUFA into phospholipids. If we had lab access, we would conduct experiments to determine the position of PUFA within phospholipids and select our phospholipase accordingly. To read more about our proposed experiments, please visit our Engineering Success page.

For a detailed, step-by-step explanation of this system for the export of AA, click here.

Phospholipase-based System for AA Export in Gram Positive Bacteria

Above: the AA export system adapted to a gram positive chassis.
  1. AA is synthesized by the A. marina PUFA synthase (Ujihara et al., FEBS Letters, 2014).

  2. AA diffuses to the inner leaflet of the cell membrane, where it embeds itself. The FakA/B3 complex phosphorylates the embedded AA to AA-PO4 (Gullet et al., J. Biol. Chem., 2019).

  3. AA-PO4 is utilized by PlsY and inserted into the cell membrane as the sn-1 component of phospholipids (Lu et al., Molecular Cell, 2006).

  4. Thraustochytrid 145138, a phospholipase capable of cleaving at the sn-1 position, cleaves phospholipids and releases free AA into the gram positive “periplasm” (Ishibashi et al., Scientific Reports, 2019).

  5. Free AA diffuses freely/passively through the cell wall.

Alternatively, it is possible that the PlsC enzyme endogenous to our potential gram positive chassis can act upon AA attached to the acyl carrier protein (ACP) of the PUFA synthase. In this case, incorporation of AA into membrane phospholipids could occur without additional engineering, and AA would be inserted at the sn-2 position of phospholipids, necessitating a PLA2 phospholipase rather than a PLA1.

Whereas DHA accumulated extracellularly in E. coli as a free fatty acid, AA was incorporated into membrane phospholipids (Metz et al., Plant Physiol. Biochem., 2009). Without additional research on heterologous expression of AA synthases within gram positive bacteria, it is unclear whether gram positive cells can incorporate the PUFA within their phospholipids. Please read our Engineering Success page to learn about how we intend to test phospholipase activity on AA and determine the appropriate phospholipid to use.

References

Gullett, J. M., Cuypers, M. G., Frank, M. W., White, S. W., & Rock, C. O. (2019). A fatty acid-binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids. The Journal of biological chemistry, 294(44), 16416–16428. https://doi.org/10.1074/jbc.RA119.010659

Hayashi, S., Satoh, Y., Ujihara, T., Takata, Y., & Dairi, T. (2016). Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins. Scientific reports, 6, 35441. https://doi.org/10.1038/srep35441

Ishibashi, Y., Aoki, K., Okino, N., Hayashi, M., & Ito, M. (2019). A thraustochytrid-specific lipase/phospholipase with unique positional specificity contributes to microbial competition and fatty acid acquisition from the environment. Scientific reports, 9(1), 16357. https://doi.org/10.1038/s41598-019-52854-7

Lu, Y. J., Zhang, Y. M., Grimes, K. D., Qi, J., Lee, R. E., & Rock, C. O. (2006). Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens. Molecular cell, 23(5), 765–772. https://doi.org/10.1016/j.molcel.2006.06.030

Metz, J. G., Kuner, J., Rosenzweig, B., Lippmeier, J. C., Roessler, P., & Zirkle, R. (2009). Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids. Plant physiology and biochemistry : PPB, 47(6), 472–478. https://doi.org/10.1016/j.plaphy.2009.02.002

Ujihara, T., Nagano, M., Wada, H., & Mitsuhashi, S. (2014). Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis. FEBS letters, 588(21), 4032–4036. https://doi.org/10.1016/j.febslet.2014.09.023

For a detailed, step-by-step explanation of this system for the export of DHA, click here.

Phospholipase-based System for DHA Export in Gram Positive Bacteria

Above: the DHA export system adapted to a gram positive chassis.
  1. DHA is synthesized as a free fatty acid by Schizochytrium PUFA synthase (Metz et al., Plant Physiol. Biochem., 2009). An additional phosphopantetheinyl transferase from Nostoc cyanobacteria is necessary for DHA synthesis when the DHA synthase is expressed heterologously.

  2. DHA diffuses to the inner leaflet of the cell membrane, where it embeds itself. The FakA/B3 complex phosphorylates the embedded DHA to DHA-PO4 (Gullet et al., J. Biol. Chem., 2019).

  3. DHA-PO4 is utilized by PlsY and inserted into the cell membrane as the sn-1 component of phospholipids (Lu et al., Molecular Cell, 2006).

  4. Thraustochytrid 145138, a phospholipase capable of cleaving at the sn-1 position, cleaves phospholipids and releases free DHA into the gram positive “periplasm” (Ishibashi et al., Scientific Reports, 2019).

  5. Free DHA diffuses freely/passively through the cell wall.



References



Gullett, J. M., Cuypers, M. G., Frank, M. W., White, S. W., & Rock, C. O. (2019). A fatty acid-binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids. The Journal of biological chemistry, 294(44), 16416–16428. https://doi.org/10.1074/jbc.RA119.010659

Ishibashi, Y., Aoki, K., Okino, N., Hayashi, M., & Ito, M. (2019). A thraustochytrid-specific lipase/phospholipase with unique positional specificity contributes to microbial competition and fatty acid acquisition from the environment. Scientific reports, 9(1), 16357. https://doi.org/10.1038/s41598-019-52854-7

Lu, Y. J., Zhang, Y. M., Grimes, K. D., Qi, J., Lee, R. E., & Rock, C. O. (2006). Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens. Molecular cell, 23(5), 765–772. https://doi.org/10.1016/j.molcel.2006.06.030

Metz, J. G., Kuner, J., Rosenzweig, B., Lippmeier, J. C., Roessler, P., & Zirkle, R. (2009). Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids. Plant physiology and biochemistry : PPB, 47(6), 472–478. https://doi.org/10.1016/j.plaphy.2009.02.002

Efflux Pump-Based AA Export System for Gram Positive Bacteria

In the absence of a β-oxidation pathway to degrade fatty acids, pathogenic bacteria such as S. aureus have two options to detoxify PUFAs secreted by the human body: 1) incorporate PUFAs into their cell membranes, or 2) efflux PUFAs. To efflux linoleic acid (LA) and AA, S. aureus utilizes an efflux pump called FarE, regulated by FarR (Alnaseri et al., J. Bacteriol., 2015; Alnaseri et al., J. Bacteriol., 2019). By removing FarE from the control of FarR and constitutively expressing it, we can constantly efflux synthesized intracellular free PUFA.

For a detailed, step-by-step explanation of this export system, click here.

  1. AA is synthesized utilizing an A. marina PUFA synthase. E. coli seems capable of activating free AA and inserting it into the membrane without further engineering (Ujihara et al., FEBS Letters, 2014). While it is unclear whether gram positive bacteria would similarly insert AA into membranes, PlsX may be able to utilize AA-ACP as a substrate, converting it to AA-PO4, which is then used for phospholipid synthesis by PlsY. PlsC may also be able to utilize AA-ACP as a substrate (Lu et al., Molecular Cell, 2006). In our model, we assume our gram positive chassis can utilize AA for membrane synthesis, and we account for a decrease in intracellular free AA that can be effluxed by FarE. In other words, the amount of free AA available is equal to AA synthesized by the PUFA synthase minus AA used by PlsX and AA used by PlsC.

  2. Free AA is pumped from the cell by FarE efflux pump (Alnaseri et al., J. Bacteriol., 2015; Alnaseri et al., J. Bacteriol., 2019).



References



Alnaseri, H., Arsic, B., Schneider, J. E., Kaiser, J. C., Scinocca, Z. C., Heinrichs, D. E., & McGavin, M. J. (2015). Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids. Journal of bacteriology, 197(11), 1893–1905. https://doi.org/10.1128/JB.02607-14

Alnaseri, H., Kuiack, R. C., Ferguson, K. A., Schneider, J. E. T., Heinrichs, D. E., & McGavin, M. J. (2019). DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in Staphylococcus aureus. Journal of Bacteriology, 201(3), e00602-00618. https://doi.org/10.1128/jb.00602-18

Lu, Y. J., Zhang, Y. M., Grimes, K. D., Qi, J., Lee, R. E., & Rock, C. O. (2006). Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens. Molecular cell, 23(5), 765–772. https://doi.org/10.1016/j.molcel.2006.06.030

Sohlenkamp, C., & Geiger, O. (2016). Bacterial membrane lipids: diversity in structures and pathways. FEMS microbiology reviews, 40(1), 133–159. https://doi.org/10.1093/femsre/fuv008

Ujihara, T., Nagano, M., Wada, H., & Mitsuhashi, S. (2014). Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis. FEBS letters, 588(21), 4032–4036. https://doi.org/10.1016/j.febslet.2014.09.023

PUFA Optimization

To optimize the probiotic’s effect on viral load, PUFA production (both AA and DHA) may be increased. Multiple strategies to boost de novo PUFA production by polyketide synthases exist. The first strategy increases PUFA production by knocking out genes related to fatty acid synthesis such as FabH (Giner-Robles et al., 2018). Deletion of these genes decreases competition between PUFA synthases and endogenous fatty acid synthases for starting substrates (such as malonyl-CoA), though it may slow the growth of the bacteria. Other strategies include the engineering of tandem ACP’s on PUFA synthases, and substitution of promoters within PUFA biosynthetic clusters with stronger, synthetic promoters (Hayashi et al., 2016; Lee et al., 2008). Finally, the introduction of foreign catalase genes may also increase PUFA production (Orikasa et al., 2007).

References



Alnaseri, H., Arsic, B., Schneider, J. E., Kaiser, J. C., Scinocca, Z. C., Heinrichs, D. E., & McGavin, M. J. (2015). Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids. Journal of bacteriology, 197(11), 1893–1905. https://doi.org/10.1128/JB.02607-14

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Docosahexaenoic 3D Conformer: PubChem Identifier: CID 445580 URL: https://pubchem.ncbi.nlm.nih.gov/compound/445580#section=3D-Conformer

Arachidonic Acid 3D Conformer: PubChem Identifier: CID 444899 URL: https://pubchem.ncbi.nlm.nih.gov/compound/444899#section=3D-Conformer