In the early stages of the project, through HP, we gained insight into the promising prospects of biopharmaceuticals in cancer therapy, prompting us to design an anticancer drug in the form of a commonly available biopharmaceutical. Our design approach involved using the Escherichia coli BL21(DE3) strain as the host cell and the J23119 strong constitutive promoter as the regulatory element to ensure sufficient production of KYNase. However, after learning about the advantages of “single-dose long-term treatment” in synthetic biology drugs through human practices, and considering the high cost of the biopharmaceutical treatment strategy, which contradicted our original goal of achieving low-cost, high-efficiency cancer treatment, the biopharmaceutical treatment plan was quickly abandoned.

We shifted our focus to designing a therapy more aligned with the innovative principles of synthetic biology, reengineering the KINETiC therapeutic approach by replacing the J23119 strong constitutive promoter with the ALPaGA responsive promoter, combined with a lysis module. The inducible promoter ensures that the engineered bacteria only release KYNase in the tumor microenvironment (TME). The lysis module acts as a timer, triggering the bacteria to lysis and release KYNase.

In the first round of DBTL, we learned from the article about an enzyme capable of significantly treating tumors—KY Nase, derived from Pseudomonas fluorescens.[1]. KYNase is a (PLP)-dependent enzyme, a member of the transaminase or α family IV subgroup, which is the largest family of PLP-dependent enzymes, with a monomeric molecular weight of approximately 45 kDa. As a characteristic of the transaminase family, KYNase monomers consist of a large domain and a small domain, with the active site located in the cleft between the domains at the subunit interface, containing residues from both subunits. KYNase exhibits excellent catalytic activity toward KYN, effectively eliminating KYN from the tumor microenvironment (TME). We obtained its sequence and sent it to a company for gene synthesis, resulting in a usable KYNase gene sequence. To ensure sustained high-level expression of our therapeutic protein, we selected Escherichia coli BL21 (DE3) as the host cell. To enable long-term efficient expression of KYNase, aiming to reduce KYN concentration and improve tumor immune suppression, we selected J23119 as the regulatory element. Parts J23100 to J23119 are a family of constitutive promoter parts from the Registry of Biological Parts (BBa_J23119). J23119 is the “consensus” promoter sequence and the strongest member of the family.

Based on these design principles, we designed and constructed the plasmid pET29a-KY Nase. We then used homologous recombination to integrate the plasmid. We transformed it into E. coli DH5α and, after picking several E. coli colonies from the transformation plates, extracted the recombinant plasmid, and performed bacterial PCR validation. The electrophoresis band position of the target fragment was correct. We transformed the plasmid into E. coli BL21(DE3) and validated the high-level expression of KY Nase through SDS-PAGE and Western blot experiments.

For the second DBTL, Zhang Gewei interviewed Professor Zhu Jingning, an expert in molecular biology. Based on the professor's advice, we decided to use the ALPaGA response promoter as a regulatory element to achieve targeted induction of engineered bacteria in a tumor environment rich in lactic acid and lacking oxygen, thereby expressing therapeutic substances. Zúñiga et al. developed a lactate-sensitive promoter called ALPaGA[2]. This promoter is sensitive to lactic-acid-rich and oxygen-deficient environments, perfect for developing a biosensor to sense colon cancer.

The replacement of the promoter ensures that our gene circuit is activated in the tumor microenvironment, preventing unintended expression of the engineered bacteria. To verify whether the ALPaGA promoter functions normally under high lactate conditions, we constructed the recombinant plasmid pET29a-ALPaGA-eGFP and introduced it into BL21(DE3) to construct the strain BL21-pET29a-ALPaGA-eGFP. We measured the eGFP expression levels of this strain at different lactate concentrations using a microplate reader to determine the optimal induction conditions for the ALPaGA promoter. As shown in the figure, we then designed and constructed the plasmid pET29a-ALPaGA-KY Nase and integrated it using homologous recombination. We transformed it into DH5α and, after inoculating several E. coli colonies from the transformation plates, extracted the recombinant plasmid and verified it by colony PCR. The colony PCR confirmed that the target fragment was correctly positioned on the gel. We transformed the plasmid into E. coli BL21(DE3) and verified the colony PCR. The BL21(DE3)-pET29a-ALPaGA-KY Nase transformation strain was successfully constructed. We performed protein expression under the optimal lactate induction concentration of the ALPaGA promoter obtained in the previous characterization and verified the high expression of KYNase through Western blot experiments.

We interviewed Wang Ruping, an expert in enzyme engineering and protein engineering, and learned that the degradation rate of kynurenine is closely related to the catalytic activity of kynureninase. Therefore, improving the catalytic activity of kynureninase is of great significance for the use of kynurenine degradation in the treatment of tumors.

In the third round of DBTL, we aim to enhance the enzymatic activity of KYNase through enzyme engineering to achieve better therapeutic outcomes. The degradation rate of kynurenine is closely correlated with the catalytic activity of KYNase. Therefore, enhancing the catalytic activity of KYNase is of significant importance for utilizing KYN degradation in tumor therapy. Semi-rational protein design is a protein engineering method that combines computational simulation and experimental validation, lying between fully rational design and directed evolution. It employs bioinformatics, structural biology, and molecular simulation techniques to purposefully modify key sites of proteins, supplemented by experimental screening and optimization to enhance protein stability, activity, specificity, or new functions. Semi-rational design first requires an analysis of the structure and function of the target protein, followed by computational-aided design, and finally the construction of mutants for experimental validation and optimization.

In this phase of the project, we engaged in more in-depth discussions with medical oncology surgeons and rectal cancer patients. We learned that the degradation and release of KYNase within the body is a critical component of the design. To enable the KY Nase produced intracellularly to be released once it reaches an effective concentration, we designed a cell lysis module based on the lytic protein PhiX174E. PhiX174E (91aa) is a protein encoded by the E gene of the phage PhiX174. Its mechanism of action is not yet fully understood, but some studies have shown that the PhiX174E protein induces cell lysis through steps involving binding to the host cell membrane, oligomerization, and proton-driven processes. First, the PhiX174E protein binds to the host cell membrane and integrates into it. Through the oligomerization of the membrane protein E, this protein can disrupt the cell membrane. This oligomerization forms a channel in the cell membrane. Once the channel is formed, the driving force for the lysis process is the osmotic pressure difference between the cytoplasm and the culture medium. Second, the E protein of the PhiX174 virus interacts with the cell's peptidoglycan, leading to its degradation and hydrolysis, thereby disrupting the integrity of the cell wall and causing limited degradation of the cell wall. Research has found that there is a time lag of approximately several minutes after the synthesis of the PhiX174E protein before cell lysis is triggered.

We utilized the ALPaGA response promoter to control the expression of PhiX174E, aiming to automatically lyse the engineered strains when the intracellular therapeutic module accumulates to a certain quantity, thereby releasing our KYNase into the tumor microenvironment for therapeutic purposes.

Based on these design principles, we designed and constructed the plasmid pET29a-ALPaGA-RBS-PhiX174E-T7 and integrated it via homologous recombination. We transformed it into DH5α and, after inoculating several single colonies of E. coli from the transformation plates, extracted the recombinant plasmid. We performed PCR validation, which confirmed the correct position of the plasmid band. We sent the plasmids with correctly positioned bands to GENEWIZ. for sequencing. As shown in the figure, the sequencing results were all correct, confirming the successful construction of the recombinant plasmid pET29a-ALPaGA-RBS-PhiX174E-T7.

Biological safety has always been a key concern for biologists. We consulted with the FDA and traditional Chinese medicine practitioners and gained some inspiration. To protect biological safety and avoid potential issues such as genetic contamination, we designed the pDawn-MazF safety module. pDawn is a blue light-induced promoter that can activate the expression of downstream genes under natural light or a single blue light source. MazF is an RNAase that can efficiently degrade bacterial mRNA, thereby inhibiting bacterial growth and reproduction.

To this end, we designed the plasmid pET-29a(+)-pDawn-MazF, as shown in the figure, to achieve the effects of self-lysis and suicide.

There are many methods to enhance enzyme catalytic activity. This experiment focused solely on modifying the stability of the protein. We can also use directed evolution to introduce random mutations into the target gene and screen for sites that enhance enzyme catalytic activity. Additionally, we can perform saturation mutagenesis on the non-conservative sequences of the enzyme-substrate binding site and screen for mutants with improved substrate binding affinity through experimental testing. Furthermore, as a biopharmaceutical for treating tumors, KYNase must function effectively within the human body. The KYNase used in this project is derived from Pseudomonas fluorescens, which may trigger an immune response in humans. In the future, we could consider modifying KYNase derived from Homosapiens to enhance its activity.

[1] Triplett TA, Garrison KC, Marshall N, et al. Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme. Nat Biotechnol. 2018;36(8):758-764. doi:10.1038/nbt.4180

[2] . Zúñiga A, Camacho M, Chang HJ, et al. Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synth Biol. 2021;10(12):3527-3536. doi:10.1021/acssynbio.1c00456