Design

This page contains the design of the wet-lab part of our project.

OVERVIEW

From 1950 to 2015, approximately 8.3 billion tons of plastics were produced globally, of which up to 79 % of waste plastics were disposed of in landfills or leaked into the environment. These large amounts of plastic gradually break down into microplastics over time. Microplastics enter oceans, lakes and other bodies of water mainly through rivers and rainwater runoff. Wastewater treatment plants are often unable to fully filter microplastics, so they also enter natural water bodies through wastewater discharges and end up in the soil as soil contaminants. These particles can damage soil structure and quality, leading to reduced crop yields and altering important properties such as weight capacity and water retention. More seriously, crops may absorb these microplastics, compromising their ability to effectively absorb nutrients and water.

To address these issues, we designed a metabolically coupled system for simultaneous degradation of microplastics and synthesis of fertilizer for the fermentation culture of Escherichia coli BL21 and Pseudomonas putida KT2440, and introduced the FAST-PETase-MHETase dual enzyme system to enable engineered E.coli to degrade polyethylene terephthalate from polymers into monomers. The tph gene cluster (tphA2-tphA3-tphB-tphA1, tpaK) was introduced for the complete uptake and utilization of terephthalic acid (TPA), and rhlA-rhlB conferred P.putida the ability to produce mono-rhamnolipids.

In order to enhance the viability of E.coli and at the same time increase the rhamnolipid production in P.putida, we also modified their metabolic pathways to some extent by overexpressing the ethylene glycol (EG) utilization pathway (fucO-aldA) in E.coli and the polyhydroxyalkanoates (PHA) degradation pathway (phaZ) in P.putida, thus optimizing the ability of the engineered bacteria to utilize different carbon sources and altering the direction of intracellular carbon flow.

Chassis strains: Escherichia coli BL21 and Pseudomonas putida KT2440

Escherichia coli BL21 (E.coli BL21) is one of the most commonly used strains for prokaryotic expression. As a well-studied chassis organism, E.coli is simple to genetically modify, easy to cultivate, and is a common chassis for the secretion of heterologous proteins. E.coli BL21 secretes PET-degrading enzymes for the degradation of PET polymers.

Pseudomonas putida KT2440 (P.putida KT2440) is a bacterium widely used in scientific research and environmental management, with a variety of unique biological properties and metabolic capabilities. KT2440 is capable of degrading a wide range of environmental pollutants, including certain plastics and organic pollutants, which makes it an important application in the field of biodegradation, and its abundant metabolic pathways can be used to biosynthesize a variety of value-added from renewable resources chemical substances 1. In addition, the genome of KT2440 was resolved in 2002, making it the first Pseudomonas putida strain to be resolved and the first Gram-negative strain to be deemed environmentally safe by the US Department of Health’s Recombinant DNA Committee 2.

Strategy 1: E.coli BL21 degradation of PET and utilization of EG

To degrade PET microplastics, we introduced a PET degradation pathway into E.coli BL21. Through this pathway, E.coli BL21 can degrade PET polymers into monomers TPA and EG, which can be absorbed and utilized by engineered bacteria.

Polyethylene terephthalate (PET) is a synthetic polyester with a high proportion of aromatic components, which is chemically inert and normally unavailable to most microorganisms. To degrade PET, we introduced FAST-PETase and MHETase to degrade PET polymers into the monomers TPA and EG.

PETase and MHETase are from the strain Ideonella sakaiensis 201-F6, and PET can be degraded by the synergistic action of the two enzymes3. FAST-PETase is a machine-learning obtained PETase with properties suitable for in situ PET degradation at mild temperatures and moderate pH conditions4.

However, the main product of PETase degradation of PET is MHET, and the MHET intermediate tends to bind tightly to PET degrading enzyme in a non-catalytic pose, which leads to the inhibition of PET degrading enzyme. Therefore, an efficient MHET hydrolase is needed to degrade the intermediate product in time to further depolymerise MHET into its monomers TPA and EG5. Multi-enzyme systems promote substrate channeling and proximity effects between enzymes. This greatly reduces the diffusion limitation between enzyme active centers, thus promoting enzyme synergy and improving catalytic efficiency6. In the process of constructing a dual enzyme system, we used bioinformatics to simulate the molecular docking of the linker connecting the two enzymes, and after simulation prediction, we chose the G4S flexible peptide as the linker of FAST-PETase and MHETase, and constructed the two into a dual enzyme system. Moreover, considering the process of constructing the dual enzyme system, the N/C-terminal arrangement of PETase/MHETase may have an impact on its overall conformation, which in turn affects its physiological activity in degrading PET. Therefore, we constructed different systems and hoped to demonstrate the difference in their activities. We hoped that E.coli could exocytosis the PETase-MHETase dual enzyme system to degrade PET microplastics in the environment. To this end, the pelB signal peptide was added to enhance the ability of BL21 to secrete PETase-MHETase 7.

Fig 1. Schematic diagram of FAST-PETase, MHETase dual enzyme system function

The final products of PET degradation by the two-enzyme system are TPA and EG. However, wild-type E.coli cannot rapidly utilize these substances for various life activities. In order to increase the efficiency of E. coli in utilizing the PET degradation products and to improve its viability, we overexpressed L-1,2-propanediol oxidoreductase and aldehyde dehydrogenase A. This modification was able to increase E.coli ‘s ability to efficiently utilize EG.

We chose fucO as the gene for L-1,2-propanediol oxidoreductase and aldA as the gene for aldehyde dehydrogenase A. L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase 8, and aldehyde dehydrogenase A is an enzyme with a relatively broad substrate specificity for small hydroxy aldehyde substrates 9. EG is first converted in E.coli to glycolaldehyde (GLA) by L-1,2 -propylene glycol oxidoreductase, which is subsequently converted to glycolic acid (GA) by aldehyde dehydrogenase A. GA can be metabolized by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of H. coli by condensing with succinate via isocitrate lyase (encoded by the aceA gene), forming isocitrate 10.

Fig 2. Diagram of EG metabolic pathway in E. coli

Strategy 2: Utilization of TPA by Pseudomonas putida KT2440

Pseudomonas putida is a common soil microorganism with abundant metabolic pathways and the ability to adapt to harsh environmental conditions and a variety of physicochemical stresses, making it a flexible and engineered metabolic engineering platform in synthetic biology 11, and the complete biosafety of the strain also makes it a common strain for environmental management and soil modification. ​
However, wild-type P.putida is unable to directly take up and utilize TPA, which is naturally possessed in the β-ketoadipic acid pathway(PCA-4,5-cleavage-pathway) in which protocatechuic acid (PCA) is a metabolic intermediate 12, and in certain strains that are able to degrade TPA based on this way, TPA can be converted to PCA. Therefore, one of the keys to the modification of KT2440 is the conversion of TPA to PCA

Fig 3. β-ketoadipic acid pathway of P.putida

P.putida KT2440 contains the introduced heterologous gene cluster (tphA2, tphA3, tphB, tphA1) from Comamonas sp.E6, which results in the conversion of P.putida intracellular TPA to PCA 13, and each of the genes in the tph gene cluster collaborates in the conversion of TPA in P.putida.

TPA 1,2-dioxygenase (TPADO) is a two-component oxygenase consisting of three parts, TphA1, TphA2, and TphA3, which together enable TPADO to effectively catalyze the oxidative reaction of TPA, converting TPA to the intermediate product 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD). TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA.TphA2, TphA3 constitute the large and small subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalyzing the oxygenation reaction in the active site.TphA2 contains the active site in direct contact with the substrate, TPA, and contains the Cys-X1-His X17-Cys-X2-His pattern, binds to Rieske-type [2Fe-2S] iron-sulfur clusters and participates in electron transfer, which is a key part of the catalytic reaction of dioxygenases , TphA3 participates in the construction of the substrate channel or the appropriate positioning of the active site, and assists TphA2 in completing the oxidation reaction; TphA1 does not directly participate in the oxygenation reaction, but it contains a [2Fe -2S] iron-sulfur cluster and a flavin adenine dinucleotide (FAD) binding site that transfers electrons from an electron donor (e.g., NADPH) to the oxidized component of TPADO 14.

Fig 4. tph gene cluster core mechanism of converting TPA to PCA

Due to the slow passive diffusion of aromatic carboxylates across the phospholipid bilayer of the bacterial inner membrane 15, we believe that the introduction of TPA transporter proteins is necessary for the transformation of P.putida.

In Comamonas sp. E6, the TPA transport system resembles a three-part tripartite tricarboxylate transporter (TTT), which consists of three components, TphC, TpiA, and TpiB, with TphC acting as a Substrate-Binding TphC specifically recognizes and binds TPA as a Substrate-Binding Protein (SBP); TpiA and TpiB are transmembrane proteins that form part of the transporter protein complex, whereas TphC delivers TPA from the periplasm (extracellular space) to these membrane proteins. Thus only when tphC and tpiA and tpiB genes are introduced simultaneously, our engineered bacteria can transport TPA 16. While TpaK, another TPA transporter protein encoded in Rhodococcus sp. RHA1, does not require the presence of tpiA or tpiB to function as a transporter, we chose TpaK as the TPA transporter protein to be applied in engineering P.putida17.

Fig 5. TPA transport pattern of TphC and TpaK

​

Strategy 3: Synthesis of rhamnolipids and pathway optimization by Pseudomonas putida KT2440

We wanted to enhance the usefulness of our system for agricultural production by producing plant-friendly products while degrading microplastic-accumulating biomass. To this end, we introduced the rhlA and rhlB genes to allow P.putida to produce mono-rhamnolipids.

Rhamnolipid is a biosurface activator first isolated from Pseudomonas aeruginosa by Jarvis and Johnson 18, Rhamnolipid emulsifies, disperses, and solubilizes hydrophobic organic pollutants and improves their bioavailability, facilitating the removal of hydrophobic organic pollutants. Rhamnolipids also have some metal chelating ability and are used to remove heavy metals from soil, sewage and other liquids. In agricultural applications, rhamnolipids can be used to improve soil, enhance the effects of pesticides and fertilizers, and inhibit agricultural diseases.

Rhamnosyltransferase I is a key enzyme necessary for the synthesis of rhamnolipids, which is a complex enzyme containing 2 subunits, RhlA and RhlB, encoded by the rhlAB gene on the same manoeuvre rhlABRI. Among them, RhlA encoded by the rhlA gene is a phthalyltransferase responsible for the synthesis of β-hydroxy fatty acids 19, whereas RhlB encoded by the rhlB gene is responsible for catalyzing the synthesis of mono-rhamnolipids containing one rhamnose group from dTDP-L-rhamnose and β-hydroxy fatty acids 20. ​
PHA are a class of biopolyesters with plastic-like properties produced by microorganisms under nutrient-limited conditions 21. P.putida synthesizes PHA via the fatty acid β-oxidation pathway and fatty acid de novo synthesis pathway, which serves as an intracellular storage material for energy and carbon sources. Whereas during PHA synthesis, it competes with rhamnolipid synthesis for the same substrate, β-hydroxyl-ACP. In order to increase the proportion of the carbon source utilized by P.putida that flows to the rhamnolipid synthesis pathway, we overexpressed the gene encoding the poly(3-hydroxyalkanoate) depolymerase gene phaZ to inhibit PHA anabolic passway and to increase rhamnolipid production.22

Fig 6. Engineered rhamnolipid synthesis pathway in P.putida

Strategy 4: Metabolic coupling of E.coli BL21 and P.putida KT2440 for degradation of microplastics in water and agricultural production

Considering the difference in growth conditions and fermentation methods between E. coli and P.putida, we referenced the previous year’s setup and separated the E.coli fermentation system from the P.putida fermentation system by using top and bottom tanks, with E.coli in the top tank being responsible for secretion of PET hydrolysis enzymes and utilizing a portion of the degradation product, EG, while P.putida was responsible for utilizing the other product of PET degradation, TPA, and the remaining EG in the bottom tank. Rhamnolipids are fermented to produce rhamnolipids, and the final rhamnolipids are sprayed into the soil to improve agricultural yields. For detailed hardware design, please refer to the Hardware.

Fig 7. Sample lab diagram of the application hardware
Click here to see the Hardware page

Pathway construction

We designed a total of three plasmids in E.coli BL21 and P.putida KT2440 respectively.

In E.coli BL21

pPeteg-P: pT7-lac operator-PETase-MHETase-T7-pT7-fucO-aldA-T7

​For FAST-PETase-MHETase, fucO, and aldA genes, we chose the T7 promoter to ensure their higher level of expression. And the lactose manipulator was added before FAST-PETase-MHETase to control gene expression.

Fig 8. schematic diagram of pPeteg-P

pPeteg-M: pT7-lac operator -MHETase-PETase -T7-pT7-fucO-aldA-T7

For MHETase-FAST-PETase, fucO, and aldA genes, we chose the T7 promoter to ensure their higher level of expression. And the lactose manipulator was added before FAST-PETase-MHETase to control gene expression.

Fig 9. schematic diagram of pPeteg-M

in P.putida KT2440

pTerephthalate: pT7- tphA2-tphA3-tphB-tphA1-T7-pT7- tpaK-T7

For both the heterologous genes tphA23B1 and tpaK, the T7 promoter was chosen to ensure their efficient expression.

Fig 10. Schematic representation of pTerephthalate-A

pRhamnolipid: pT7-lac operator-rhlA-rhlB-T7-pT7-lac operator-phaZ-T7

Both use the T7 promoter and lac operator to express rhlAB and phaZ to control the production of rhamnolipids by P.putida.

Fig 11. schematic representation of pRhamnolipid

References

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