Introduction

Every iGEM team starts by determining their project's focus. While our aim was to excel in the iGEM competition, we also wanted to contribute to the synthetic biology community and our local area. We often see plastic waste in our town and nearby woods, and this issue is prevalent globally. This led us to explore plastic degradation using Synthetic Biology. While we don't endorse continued plastic pollution, our method provides an eco-friendly solution to existing plastic waste. We've devised a cost-effective, sustainable way to break down PET, a widespread plastic. Using two enzymes, Fast-PETase and MHETase, we can convert PET into its basic components: ethylene glycol and terephthalic acid.

We center our efforts on sewage treatment facilities (see the implementation), which requires us to select an appropriate chassis that can thrive and produce our enzyme within the system.

Chassis Selection

In Synthetic Biology a chassis refers to the cellular host or organism that is used as a platform to introduce and express synthetic genetic circuits or pathways. The chassis provides the necessary cellular machinery and environment for the synthetic components to function. Commonly used chassis organisms include bacteria like Escherichia coli and yeast like Saccharomyces cerevisiae. Using microalgae as a chassis in our project, especially for applications in sewage treatment facilities, offers several advantages over commonly used organisms like Escherichia coli or Saccharomyces cerevisiae: photosynthesis, carbon sequestration, nutrient uptake, tolerance to harsh conditions and reduced pathogenic concerns. To choose the best microalgae as a chassis, we carried out a comparison between different species:

The choice of chassis is crucial because it can influence the efficiency, scalability, and overall success of the Synthetic Biology project. The ideal chassis is often one that is well-understood, easy to genetically manipulate, and can be grown under controlled conditions. In our research group, we choose to utilize Chlamydomonas reinhardtii as our chassis organism. It's a preferred choice for producing recombinant proteins, and there are investigations that demonstrate the feasibility of expressing wild type PETase in this species of microalgae. Due to its ease of cultivation and handling, C. reinhardtii does not form biofilms; the absence of biofilm formation means that the microalga will remain suspended in the culture medium, making it easier to harvest and process.

We initially chose to work with the CC400 strain, a variant in molecular biology due to its absence of a cell wall. This characteristic facilitates easier transmembrane transport and secretion of proteins and other molecules. Additionally, the absence of a cell wall can simplify transformation procedures, such glass beads. This strain's unique properties provide a valuable platform for advanced molecular biology research and applications.

Expression of Recombinant Proteins in the Nucleus or Chloroplast?

In the realm of molecular biology and biotechnology, the transformation of organisms to introduce foreign genes is a foundational technique. Two primary sites for introducing these genes in eukaryotic cells, especially in plants and certain algae, are the nucleus and the chloroplast. Each location offers its own set of advantages and challenges, making the choice between them pivotal for the success of the experiment. While nuclear transformation involves the integration of foreign DNA into the host's nuclear genome, chloroplast transformation targets the DNA within the chloroplasts. This comparison delves into the intricacies of both methods, highlighting their respective merits, limitations, and ideal applications. As researchers aim to harness the full potential of genetic engineering, understanding the nuances between nuclear and chloroplast transformation becomes paramount. That’s why we do a comparison between both:

We choose to express our recombinant proteins in the nucleus of C. reinhardtii. Expressing recombinant proteins in the nucleus of C. reinhardtii offers a strategic advantage, primarily due to the capability for protein secretion. This approach simplifies purification, ensures proper protein modifications, and facilitates large-scale production. By leveraging the alga's natural secretion pathways, we can achieve efficient, high-quality protein yields, making C. reinhardtii a valuable host for our implementation.

Enzymes that degrade PET

In the intricate world of molecular biology, enzymes play a pivotal role, acting as biocatalysts that drive countless biochemical reactions. Their specificity, efficiency, and adaptability make them invaluable tools in both natural biological processes and biotechnological applications. Among the vast array of enzymes, wild PETase, Fast-PETase, MHETase and PHL7 have garnered attention in our design for their properties and applications.

As we delve into the comparative analysis of these enzymes, the overarching goal is to understand their potential in biodegrading plastics and aiding the bioremediation of water bodies. By harnessing the power of these enzymes, we stand a chance to reverse the damage caused by microplastics and restore the health of our aquatic ecosystems. Below are 4 enzymes that we analyzed for use in the project:

PETase

PETase is an enzyme identified in the bacterium Ideonella sakaiensis 201-F6. This enzyme is capable of hydrolyzing poly(ethylene terephthalate) (PET), a common plastic material that is resistant to microbial degradation. PETase exhibits a distinct preference for PET over other substrates. It efficiently hydrolyzes PET, producing mono(2-hydroxyethyl) terephthalic acid (MHET) as the major product. PETase has prominent hydrolytic activity for PET, especially in its glassy state, which is critical for the growth of I. sakaiensis on PET in various environments. PETase also exhibits higher activity against commercial bottle-derived PET, which is highly crystallized. I. sakaiensis adheres to PET and secretes PETase to target this material. The exact binding mechanism of PETase is not detailed, but it's noted that without a three-dimensional structure determined for PETase, the exact binding mechanism remains unknown. The discovery of PETase offers a potential biological solution for PET degradation. The enzyme's ability to break down PET into its constituent monomers can pave the way for environmentally friendly remediation strategies.

Fast-PETase

FAST-PETase is a mutant variant derived from the PETase enzyme. It contains five mutations compared to the wild-type PETase. These mutations include N233K, R224Q, and S121E from prediction, along with D186H and R280A from the scaffold. This enzyme demonstrated superior PET-hydrolytic activity relative to both wild-type and other engineered alternatives between 30 and 50°C across a range of pH levels. At 50°C, FAST-PETase showed the highest overall degradation of all mutants tested, releasing 33.8 mM of PET monomers (the sum of terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalate (MHET)). The enzyme's activity against post-consumer PET (pc-PET) was substantially higher than that of other enzymes like WT PETase, ThermoPETase, DuraPETase, LCC, and ICCM under the same conditions. FAST-PETase was able to almost completely degrade untreated post-consumer PET from 51 different thermoformed products in just one week. The enzyme also demonstrated the capability to depolymerize untreated, amorphous portions of a commercial water bottle. A time-course analysis revealed an almost linear decay rate of PET degradation and a concomitant increase in crystallinity over time. The development of FAST-PETase offers a potential solution for the degradation of PET plastics, especially given its enhanced activity and stability across a range of conditions. This enzyme can play a pivotal role in addressing the environmental challenges posed by PET plastic accumulation.

MHETase

MHETase is an enzyme identified in the bacterium Ideonella sakaiensis. It is responsible for the hydrolysis of MHET (mono(2-hydroxyethyl) terephthalic acid). This enzyme hydrolyzes MHET to produce TPA (terephthalic acid) and EG (ethylene glycol). The gene encoding MHETase in I. sakaiensis is designated as ISF6_0224. This gene is located adjacent to the TPA degradation gene cluster. The ISF6_0224 protein sequence matches those of the tannase family, which is known to hydrolyze the ester linkage of aromatic compounds such as gallic acid esters, ferulic saccharides, and chlorogenic acids.The purified recombinant ISF6_0224 protein efficiently hydrolyzed MHET with a turnover rate (kcat) of 31 ± 0.8 s⁻¹ and a Michaelis constant (Km) of 7.3 ± 0.6 mM. The enzyme did not show any activity against PET, BHET, pNP-aliphatic esters, or typical aromatic ester compounds catalyzed by the tannase family enzymes. The discovery of MHETase in I. sakaiensis provides insight into the metabolic pathway of PET degradation by this bacterium. The enzyme plays a crucial role in breaking down PET into its simpler constituents, which can then be further metabolized by the bacterium. In our design we use BBa_K3002037 the basic part from Kaiser Collection, this part contains the introns 1 and two times intron 2 of RBCS2, that perfectly matches to pAR promoter.

PHL7

PHL7 is a recently discovered metagenomic-derived polyester hydrolase. This enzyme is capable of efficiently degrading amorphous polyethylene terephthalate (PET) found in post-consumer plastic waste. It Is stable in a temperature range suitable for PET hydrolysis between 65°C to 70°C. It hydrolyzes PET most effectively around 70°C, which is close to the glass transition temperature (Tg) of the polymer. PHL7 interacts with the terephthalic acid (TPA) moiety of its substrate in a lock-and-key mechanism, rather than an induced fit. This is similar to the enzyme LCC but different from IsPETase. The ligand-induced opening of the substrate-binding groove in PHL7 is restricted due to decreased flexibility of subsite I, which is influenced by the residue H185. This prevents the aromatic residue W156 from moving away from the active site. The aromatic π-stacking clamp, composed of residues F63 and W156, is crucial for the binding and hydrolysis of BHET. The overall folds of PHL7 and its cocrystal structure with TPA (PHL7×TPA) are nearly identical, with minor deviations in the orientations of active site amino acids.

Our Design

Simultaneously with the search for the selection of our chassis, which ultimately was C. reinhardtii, we designed our genetic circuit. The main objective of this circuit is to include the genetic information to produce and secrete mainly two enzymes, Fast-PETase and MHETase, with the purpose of degrading Polyethylene Terephthalate (PET).

Initially, our design idea was to make several constructions and assemble them using the Modular Cloning system. However, based on our context it seemed more efficient to do it through Gibson Assembly. The first circuit designs were the ones shown below, but they were eventually discarded. If you know why we discarded it, see the engineering success.

After discarding the circuits above, we made 4 designs of genetic circuits. These genetic circuit sequences were validated by our Principal Investigator Cristhian Rojas and our advisor João Molino and aims to express and secrete our enzymes of interest. In this base sequence, we varied the Enzyme region to incorporate our enzymes of interest, Fast-PETase and MHETase."

Genetics circuits

Summary of the 4 genetic circuits. If you want to know more about the different parts of the circuit, you can look in the parts section of this page.

Circuit number 1, with the PAR promoter, the bleomycin/zeocin resistance sequence, signal peptide, the Fast-PETase, and the terminator. Designed to express and secrete Fast-PETase in Chlamydomonas reinhardtii, utilizing pJP32 backbone.

Circuit number 2, with the PAR promoter, the bleomycin/zeocin resistance sequence, signal peptide, the Fast-PETase linked to MHETase, Tag, and the terminator. Designed to express and secrete Fast-PETase linked to MHETase in Chlamydomonas reinhardtii, utilizing pJP32 backbone.

Circuit number 3, with the PAR promoter, the bleomycin/zeocin resistance sequence, signal peptide, the Fast-PETase, gene reporter mCherry, and the terminator. Designed to express and secrete Fast-PETase with verification by mCherry reporter in Chlamydomonas reinhardtii, utilizing pJP32 backbone.

Circuit number 4, with the PAR promoter, the bleomycin/zeocin resistance sequence, signal peptide, the PHL7 enzyme and the terminator. Designed to express and secrete PHL7 in Chlamydomonas reinhardtii, utilizing pJP32 backbone. This circuit is originally designed by our advisor João Molino.

Parts

We chose the PAR promoter because it is a promoter sequence commonly used in Chlamydomonas reinhardtii for the expression of transgenes. The promoter in question is light-inducible, and its activity is modulated by the availability of light-harvesting complexes, specifically the phycobilisome complex. PAR is relatively strong, and its expression can be adjusted by changing the intensity and duration of light exposure. This promoter is a combination of the HSP70A (upstream) and RBCS2 promoters; no heat shock is required as a transcription activator. Several characteristics of this promoter suggest its usefulness as a tool to enhance transgene expression in this alga. It can, by itself, confer high inducibility to a transgene.

Reference: Schroda, M., Blöcker, D., & Beck, C. F. (2000). The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant Journal, 21(2), 121-131. https://doi.org/10.1046/j.1365-313x.2000.00652.x

The bleomycin resistance gene confers resistance to the antibiotic bleomycin and zeocin. In the context of Chlamydomonas reinhardtii the bleomycin resistance of cells harboring the ble gene is attributed to the action of the bleomycin-binding protein. This protein binds to bleomycin, sequestering it and preventing it from exerting its antibiotic effect on the cell. This resistance mechanism is particularly useful in genetic engineering experiments. By introducing the ble gene alongside other genes of interest into Chlamydomonas reinhardtii, researchers can easily select for cells that have taken up the foreign DNA by exposing them to bleomycin. Only the cells that have incorporated the ble gene (and by extension, the other genes of interest) will survive, allowing for efficient selection.

Reference: Gatignol, A., Durand, H., & Tiraby, G. (1988). Bleomycin resistance conferred by a drug‐binding protein. FEBS Letters, 230.

For the constructs expressed in Chlamydomonas reinhardtii, we used the signal peptide SP7 which has been recently characterised in a paper by João Molino. SP7 is a novel protein identified in the flagella of Chlamydomonas reinhardtii. The flagella are whip-like appendages that protrude from the cell body and are vital for motility and sensory functions. Which confers an interesting capacity to secrete recombinant proteins.

Reference: Molino JVD, de Carvalho JCM, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii. PLoS ONE 13(2): e0192433. https://doi.org/10.1371/journal.pone.0192433

FAST-PETase is derived from the original sequence of the PETase enzyme, with several modifications introduced through directed evolution. The FAST-PETase sequence is approximately 590 amino acids in length and consists of several domains, including a signal peptide sequence for secretion (twin-arginine translocation (Tat) signal peptide), a catalytic domain, and a carbohydrate-binding module. The catalytic domain is responsible for breaking the ester bonds in PET, while the carbohydrate-binding module helps anchor the enzyme to the plastic surface. Directed mutations have enhanced the enzyme's ability to bind to PET and break it down into its components more quickly. Therefore, we chose FAST-PETase because it is capable of breaking down PET at a faster rate than the original PETase enzyme.

Reference: Lu, H., Diaz, D.J., Czarnecki, N.J. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022). https://doi.org/10.1038/s41586-022-04599-z

Linker, the most basic function of linkers in recombinant proteins is to covalently join functional domains (e.g., flexible linkers or rigid linkers) or release them under desired conditions (clickable linkers). Linkers can also provide many derived functions, such as improving the folding and stability of recombinant proteins, enhancing the expression of fusion proteins, and they can enhance the bioactivity of fusion proteins. The linker we chose binds to the C-terminal region of MHETase to the N-terminal of PETase, with flexible glycine-serine linkers of 12 total residues (Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly). The following image graphically shows how a linker is composed, with glycine in orange and serine in yellow:

Reference: KNOTT, B. C. et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of the United States of America, v. 117, n. 41, p. 25476–25485, 13 out. 2020. https://doi.org/10.1073/pnas.2006753117 CHEN, X.; ZARO, J. L.; SHEN, W.-C. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, v. 65, n. 10, p. 1357–1369, out. 2013. http://dx.doi.org/10.1016/j.addr.2012.09.039

MHETase is an enzyme capable of breaking down MHET into its monomers, terephthalic acid (TA) and ethylene glycol (EG), through the cleavage of the ester bond. The enzyme is composed of two domains: a large N-terminal domain and a small C-terminal domain, connected by a linker region. The N-terminal domain contains the enzyme's catalytic site and is responsible for substrate recognition and binding, while the C-terminal domain plays a role in regulating the enzyme's activity.

Reference: Maity, W., Maity, S., Bera, S. et al. Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Appl Biochem Biotechnol 193, 2699–2716 (2021). https://doi.org/10.1007/s12010-021-03562-4

PHL7 is a thermophilic polyester hydrolase isolated from a plant compost metagenome along with six homologs (PHL1-6)4. It rapidly hydrolyzes amorphous PET at 70°C, producing terephthalic acid (TPA) and ethylene glycol (EG), outperforming all previously reported PET hydrolytic enzymes, including modified variants.

Reference: RICHTER, P. K. et al. Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product. Nature Communications, v. 14, n. 1, p. 1905, 5 abr. 2023. https://doi.org/10.1038/s41467-023-37415-x

RPL23 is a termination signal used to terminate the transcription of the RPL23 gene. This sequence signals the RNA polymerase to dissociate from the DNA template and release the newly synthesized RNA molecule. The RPL23 terminator sequence has been found to work well in terminating transcription in a variety of organisms and is often used as a standard terminator sequence in genetic engineering.

Reference: LÓPEZ‐PAZ, C. et al. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. The Plant Journal, v. 92, n. 6, p. 1232–1244, 18 nov. 2017. https://doi.org/10.1111/tpj.13731

mCherry is a popular fluorescent protein derived from Discosoma sp. and is frequently used as a reporter gene in various organisms due to its bright red fluorescence. In the context of Chlamydomonas reinhardtii, a model green alga, mCherry has been employed as a valuable tool for genetic engineering. The expression level of mCherry can be correlated with the activity of promoters or other genetic elements, aiding in the analysis of gene expression patterns in Chlamydomonas.

Reference: ARIAS, C. et al. Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii. Biotechnology Progress, v. 37, n. 2, 20 nov. 2020.

Gibson Assembly HiFi

Gibson Assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. Developed by Dr. Daniel Gibson and his colleagues, this method has become a popular alternative to traditional cloning techniques due to its efficiency and simplicity. Now we gonna explaining what it is and how to use it:

What is Gibson Assembly?

• Isothermal Assembly: Unlike other methods that require temperature cycling, Gibson Assembly can be performed at a constant temperature, typically 50°C.
• Joining Multiple Fragments: It allows for the simultaneous and seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility.
• Enzymatic Reaction: The method uses a mix of three enzymes: an exonuclease, a DNA polymerase, and a DNA ligase. These enzymes work in tandem to join the DNA fragments.

How to Use Gibson Assembly?

• Design Overlaps: Begin by designing overlapping sequences between adjacent DNA fragments. Typically, overlaps are 20-40 base pairs long.
• Prepare DNA Fragments: DNA fragments can be generated using PCR, ensuring that the ends of the PCR products have the necessary overlaps.
• Mix Fragments: Combine the DNA fragments in a single tube.
• Add Gibson Assembly Master Mix: This mix contains the three enzymes required for the assembly. It's commercially available, making the process straightforward.
• Incubate: Incubate the reaction mixture at 50°C for about an hour.
• Transformation: Once the assembly is complete, the resulting DNA can be introduced into bacterial cells (e.g., E. coli) using standard transformation techniques.
• Verification: After transformation, it's essential to verify the correct assembly using methods like colony PCR or sequencing.

We choose Gibson Assembly to assemble our genetics circuits because this methodology offers efficiency, so we can assemble multiple fragments in a single step, flexibility because works with fragments of varying lengths and end compatibilities, and speed since the entire process can be completed in a few hours.

Future Challenges

• Heterologous Gene Expression in Chlamydomonas Reinhardtii Heterologous genes introduced into the nuclear genome of Chlamydomonas Reinhardtii are often poorly expressed. This can pose a challenge for the effective expression of PETase and MHETase in this microalga.

• Enzyme Structure and Function: MHETase breaks down MHET into its monomers, terephthalic acid (TA) and ethylene glycol (EG). The enzyme has a complex structure with multiple domains, and any alteration or malfunction, like use a linker in these domains can affect its activity.

• Fast-PETase Specificity: The Fast-PETase enzyme has been designed to break down PET at a faster rate than the original PETase enzyme. However, its specificity and efficiency in real-world water treatment scenarios, especially in the presence of other contaminants, remain a challenge.

• Implementation in Sewage Treatment Facilities: Introducing and maintaining a genetically modified organism like Chlamydomonas Reinhardtii in such facilities, ensuring its survival, and preventing any unintended ecological consequences is challenging.

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