Indole
Pathway Design
Research has shown that GDIAS-5 demonstrates robust indole degradation potential, achieving an indole degradation rate of 93.7±5.4% after a 24-hour cultivation period. Research underscores the native ycnE enzyme's efficacy in converting indole to isatin. Simultaneously, FMO enzymatically converts indole to indoxyl, which subsequently undergoes spontaneous oxidation to indigo in aerobic conditions. Investigations illustrate that introducing FMO into E.coli and culturing for 24 hours can yield a maximum of 400 mg/L indigo[1]. Furthermore, E.coli itself constitutes a source of indole. The tnaA enzyme, endogenously expressed by E.coli, catalyzes the conversion of tryptophan to indole, representing a principal source of E.coli 's characteristic malodor. To effectively mitigate the malodorous emanations arising from indole, we have chosen to disrupt E.coli 's endogenous pathway for indole production. This involves the targeted knockout of the tnaA gene within E.coli through the λ-Red homologous recombination system, yielding an indole-free strain of E.coli. Subsequently, we will proceed to engineer this strain by introducing genes encoding both ycnE and FMO enzymes. This dual strategy aims to not only abrogate the inherent malodor of E.coli but also achieve effective degradation of exogenous indole.
Vector Construction
We acquired gene sequences associated with indole degradation and the tnaA enzyme from the National Center for Biotechnology Information (NCBI) and independently devised the working plasmid piGEM23_04. Post-plasmid synthesis, we intend to conduct Polymerase Chain Reaction (PCR) and first-generation sequencing to ascertain the plasmid's correct assembly and detect potential point mutations.
Nicotine
Pathway Design
Research indicates that Pseudomonas putida S16, utilizing the Pyrrolidine pathway, achieves a nicotine degradation rate of 4.0g/13h[2]. Additionally, Tang et al. demonstrated successful incorporation of a partial nicotine degradation gene cluster from Pseudomonas putida S16 into E.coli DH5α, conferring efficient nicotine degradation capabilities[3,4].
Leveraging these insights, we designed and integrated the complete nicotine degradation gene cluster into a functional plasmid, subsequently introducing it into E.coli Top10 to optimize "degradation + utilization" effects.
Vector Construction
We obtained the gene sequences of the nicotine degradation gene cluster from NCBI. We opted for employing pUC57Simple and pUSP vectors carrying the nicotine degradation gene cluster as the backbone. Enzymatic cleavage was conducted using the BsmBI restriction endonuclease, followed by homologous recombination assembly through Gibson, resulting in the construction of the functional plasmid, piGEM23_01.
Benzo[a]pyrene
Pathway Design
To counter the dangers of benzo[a]pyrene in second-hand smoke, we integrated Bacillus subtilis' catechol oxidase cotA and Pseudomonas putida's catechol 1,2-dioxygenase catA into E.coli, engineering it to degrade benzo[a]pyrene. CotA initiates the degradation by oxygenating benzo[a]pyrene's carbon rings, forming quinone-like intermediates, more easily degradable due to higher water solubility and weaker phenolic structures. Further degradation of the quinone-like compounds is facilitated by catechol 1,2-dioxygenase from Pseudomonas putida, inducing ortho-cleavage of catechol, a phenolic intermediate. 1,2-dioxygenase's role in BaP degradation was previously established, justifying its inclusion in the proposed PAH metabolic pathway. Optimal conditions for catechol 1,2-dioxygenase are pH 7.5-8 and 25-30°C, aligning with experimental requirements. However, the intermediate quinone, although harmless, prompts E.coli self-destruction, hindering further transformation. To address this, we identified the quinone sensing and response inhibitor (QsrR) through literature review. QsrR, from Staphylococcus aureus, selectively binds to palindrome DNA sequences, blocking RNA polymerase and inhibiting transcription. In the presence of quinones, it reshapes, initiating transcription by binding and dissociating from target DNA. As CotA initially degrades PAHs into quinone, these intermediates act as signaling molecules, inducing expression of enzymes for subsequent degradation. This approach conserves energy for cells expressing these PAH-degrading enzymes[5].
![The Benzo[a]pyrene degradation pathway](https://static.igem.wiki/teams/4773/wiki/pageimage/pageimage/design-the-benzo-a-pyrene-degradation-pathway.png)
Vector Construction
We acquired the gene cluster sequences associated with benzo[a]pyrene degradation from NCBI. Our design involved plasmids pUC57Simple_cotA, pUC57Simple_catA, and pUSP, each containing BsmBI restriction sites, serving as the structural foundation for this segment. Utilizing enzymatic cleavage and Gibson assembly, we successfully synthesized the functional plasmid, piGEM23_02.
![The plasmid of benzo[a]pyrene degradation pathway](https://static.igem.wiki/teams/4773/wiki/pageimage/pageimage/design-the-plasmid-of-benzo-a-pyrene-degradation-pathway.png)
Formaldehyde
Pathway Design
Our project aims to confer formaldehyde degradation capability uponE.coli. Our strategy involves expressing Methylobacillus flagellatus genes, Hps and Phi, encoding 6-phospho-hexose isomerase and 6-phospho-3-hexose isomerase, respectively, in E.coli Top10. This expression establishes the ribulose monophosphate (RuMP) pathway, facilitating formaldehyde assimilation. Additionally, E.coli 's frmA gene encodes a glutathione-dependent formaldehyde dehydrogenase, catalyzing the conversion of formaldehyde to formate. Knocking out frmA redirects formaldehyde towards assimilation rather than dissimilation, converting it into metabolically utilizable substances[6].
To optimize RuMP pathway performance, we focus on ribulose-5-phosphate (Ru5P) production, a crucial intermediate in formaldehyde assimilation.E.coli primarily utilizes the glycolytic pathway for glucose metabolism, resulting in limited carbon flux through the pentose phosphate pathway, hence affecting Ru5P levels. To address this, we plan to knock out the pgi gene, responsible for glucose-6-phosphate isomerization in the glycolytic pathway. This genetic modification redirects carbon flux towards the pentose phosphate pathway, favoring Ru5P production[7].
In summary, the introduction of hps and phi genes enables formaldehyde assimilation via the RuMP pathway in E.coli, converting formaldehyde to fructose-6-phosphate (F6P) and further metabolizing it into acetyl-CoA, ultimately entering the tricarboxylic acid cycle. Simultaneously, by knocking out frmA and pgi genes, we enhance the formaldehyde assimilation pathway and promote Ru5P generation through the pentose phosphate pathway.
Vector Construction
We obtained the gene sequences for hps and phi from the National Center for Biotechnology Information (NCBI) and designed plasmid piGEM23_03 accordingly. Our strategy entails employing λ-Red homologous recombination to effectuate the knockout of frmA and pgi genes. Following positive clone identification, we will validate the gene knockout outcomes via Polymerase Chain Reaction (PCR). Subsequently, plasmid piGEM23_03, constructed utilizing enzymatic digestion and Gibson assembly, will be transformed into E.coli.
Butyric Acid
Pathway Design
We targeted three crucial enzymes from Acetobacter butylicus for butyric acid degradation: ptb, buk, and adhE2. Literature indicates that integrating these enzymes can yield 1.7 mM butanol from 2.3 mM butyric acid, showcasing high efficiency in butyric acid degradation[8]. Additionally, we introduced ATF1 enzyme from Saccharomyces cerevisiae s288c to facilitate product reuse, generating fragrant acetyl butyrate. Research affirms the production of approximately 0.39±0.01g/L of acetyl butyrate using this enzyme[9].
Innovatively, we combined the butyric acid degradation pathway, producing butanol, with the butanol esterification pathway. Integrating these four crucial enzymes into the working plasmid, we aim to introduce the plasmid into our chassis organism, E.coli Top10, anticipating the achievement of a "deodorization + fragrance production" effect.
Vector Construction
We acquired the gene sequences related to butyric acid degradation from NCBI and devised plasmids pUC57Simple_buk_ptb and pUC57Simple_adhE2, both incorporating the BsmBI restriction sites. Concurrently, to achieve deodorization and fragrance production through ester compounds, we designed pUC57Simple_ATF1, also featuring the BsmBI restriction site, to synthesize lipid-like substances from degradation byproducts. Employing pUSP as the foundational framework, we conducted enzymatic cleavage and Gibson assembly, culminating in the synthesis of the functional plasmid, piGEM23_05.
Hydrogen Sulfide
Pathway Design
Most organisms possess metabolic pathways that reduce sulfates to hydrogen sulfide. However, pathways for oxidizing hydrogen sulfide are less common and are primarily found in sulfur bacteria and other chemolithoautotrophic bacteria. We referenced Tongji University's 2021 iGEM project and selected four genes: SQR, SDO, AprBA, and SAT, to construct a gene expression vector, which we introduced into E.coli. This modification equips the bacteria with the ability to oxidize hydrogen sulfide into non-toxic sulfate.
Vector Construction
We obtained gene sequences related to hydrogen sulfide degradation from NCBI. Our design involved plasmids pUC57Simple_SQR_SDO, pUC57Simple_AprBA_SAT, both incorporating the BsmBI restriction sites, and pUSP, serving as the foundational framework. Through enzymatic cleavage and Gibson assembly, we successfully synthesized the functional plasmid, piGEM23_06.
Ammonia
Pathway Design
Drawing inspiration from Tongji University's 2021 iGEM project, we selected AMO, HAO, and NOD enzymes to facilitate the transformation of ammonia to nitrate. After E.coli absorbs ammonia, AMO converts it to hydroxylamine. Subsequently, externally introduced hydroxylamine oxidoreductase HAO facilitates the transformation from hydroxylamine to nitric oxide, and finally, nitric oxide is further oxidized to nitrate by nitric oxide dioxygenase HmpA.
Vector Construction
We obtained gene sequences related to ammonia degradation from NCBI. Our design included plasmids pUC57Simple_HAO and pUC57Simple_HmpA, both featuring the BsmBI restriction sites, along with pUSP as the foundational framework. Utilizing enzymatic cleavage and Gibson assembly, we successfully synthesized the functional plasmid, piGEM23_07.
Bacteria Immobilization
In order to increase the concentration of engineering bacteria in the bioreactor and ensure the activity of the engineering bacteria without leaking into the environment, the engineering bacteria need to be fixed. In recent years, hydrogels have been considered as ideal materials for immobilization due to their good adsorption capacity, degradability and biocompatibility. Therefore, we chose the hydrogel embedding method to immobilize the engineered bacteria.
Sodium Alginate
First, we chose to use the reaction of sodium alginate and calcium chloride to embed engineering bacteria to make sodium alginate microbeads. Sodium alginate is a natural polysaccharide carbohydrate that is widely used due to its low price, good hygroscopicity and easy degradation[10]. The preparation method of sodium alginate microbeads is simple, low-cost, mature in application, and suitable for commercial use.
PNIPAm Photoreactive Hydrogel
In addition to sodium alginate embedding, we are also actively exploring some new immobilization methods. In recent years, poly-N-isopropylacrylamide (PNIPAm) hydrogel has become one of the hot spots of research and has attracted widespread attention due to its endothermic shrinkage characteristics. At the same time, according to previous research, adding dopamine, which absorbs light energy, can make a photoreactive hydrogel, allowing a certain degree of control over biological reactions. Therefore, we also tried to make PNIPAm photoreactive hydrogel to explore a more complete and efficient immobilization method.
