This study selected the plasmid pET-29a (+) -KYNase that has been successfully constructed in the laboratory as the research target. As shown in Figure 1-1-1. We selected the J23119 strong constitutive promoter and Escherichia coli BL21 (DE3) as the promoter element and chassis cells, respectively, to enable long-term and efficient expression of KYNase. On this basis, we will isolate and purify the KYNase expressed by the engineered strain we constructed and use it as a biological preparation.

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-KYNase. 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.

1: Marker (Takara); 2-3: PCR-KYNase

1: Marker (Takara); 2-24: PCR-kynase; 25: Marker (Takara)

Sequencing results of plasmid pET29a-KYNase Company

We first characterized the KYNase containing a constitutive . We extracted the correctly sequenced plasmid pET-29a(+)-KYNase and transfected it into E. coli BL21(DE3) competent cells for expression. In the experiment, single colonies were selected and cultured in LB culture medium containing the resistance gene Kana and divided into experimental group and control group. After the bacterial solution OD600.value is close to 1.0. Set the induction temperature to 37°C and the induction time to 14 hours. Place the bacterial solution in a 50 mL centrifuge tube and centrifuge to precipitate. Discard the supernatant, add buffer to resuspend the precipitate, and then perform ultrasonic disruption. After ultrasonic disruption, centrifuge, collect the supernatant, resuspend the precipitate, and save the sample. As shown in Figure 1-2-1, lanes 1, 3, 5, and 7 are our blank control bands, and lanes 2, 4, 6, and 8 are our experimental bands. We will focus on lanes 5 and 6. We set the supernatant of the blank control group after crushing and centrifugation as the 5th lane, and the supernatant of the experimental group after crushing and centrifugation as the 6th lane. It can be clearly seen that compared with lane 5, lane 6 has an obvious band around 40 kDa. Moreover, before and after ultrasonic disruption, there was an obvious band at about 40 kDa in the experimental group and in the precipitate resuspension, which was not found in the blank control group. Therefore, we preliminarily determined that kynurenine enzyme was successfully expressed in E. coli BL21 (DE3). The same bands appeared in the precipitate resuspension of the experimental group, which may be due to the fact that part of the target protein was not folded correctly and formed inclusion bodies.

MMarker1Blank control group before ultrasonic disruption (E. coli BL21(DE3) with pET-29a(+) plasmid)2Experimental group before ultrasonic disruption (E. coli BL21(DE3) with pET-29a(+)-KYNase)3Crude enzyme solution of blank control group after ultrasonic disruption4Crude enzyme solution of the experimental group after ultrasonic disruption5Blank control group supernatant6Experimental group supernatant7Blank control group precipitate resuspension8Experimental group precipitation resuspension.

The protein samples stored in the above experiment were spotted in the same order, and the membrane was transferred after protein electrophoresis. The transferred membrane was blocked with a quick blocking solution and then incubated with the primary antibody overnight. After washing the membrane, the secondary antibody was incubated immediately, and finally, the developer was dripped on for development and the results were observed. The development results are shown in Figure 1-2-2. The internal reference band is clearly visible, indicating that the sample processing, electrophoresis, and membrane transfer steps in the experiment were properly performed, and the experimental quality was high. Comparing the blank control group with the experimental group, it can be clearly seen that there are bands in the 2nd, 4th, 6th, and 8th lanes, but not in the 1st, 3rd, 5th, and 7th lanes. The experimental results of Western Blot were consistent with those of SDS-Page, both indicating that the kynurenine enzyme was successfully expressed.

1Blank control group before ultrasonic disruption (E. coli BL21 (DE3) with pET-29a (+) plasmid)2Experimental group before ultrasonic disruption (E. coli BL21(DE3) with pET-29a(+)-KYNase)3Crude enzyme solution of blank control group after ultrasonic disruption4Crude enzyme solution of the experimental group after ultrasonic disruption5Blank control group supernatant6Experimental group supernatant7Blank control group precipitate resuspension8Experimental group precipitation resuspension.

In order to verify whether the ALPaGA promoter can function normally under high lactic acid conditions, the recombinant plasmid pET29a-ALPaGA-eGFP was constructed to verify the function of the ALPaGA promoter by expressing eGFP (green fluorescent protein). Pick a single colony of BL21-pET29a-ALPaGA-eGFP from the streaked plate and culture it. Add 5uL 100g/L KanR to 5 ml LB liquid medium, mix well, and culture overnight at 37℃ and 220rpm in a shaking incubator for 12-16h. Take 1 ml of overnight cultured bacterial solution and add it to 5 bottles of 50 ml LB liquid culture medium to dilute the bacterial solution to OD600 = 0.1. Culture at 37°C, 220 rpm for 1-2 h. When OD600 = 0.6-0.8, add 1*10-3M, 1*10-2M, 1*10-1M, and 1M L-lactic acid to 4 shake flasks respectively. Induce overnight at 37°C, 220 rpm for 12 h.

Take 1mL of bacterial solution from each group after overnight culture and centrifuge at 5000rpm for 10min, discard the culture supernatant, wash with PBS and resuspend the centrifuged bacterial slurry, repeat this operation twice. Pipette 200ul of bacterial solution (after resuspending in PBS) from each group onto a 96-well plate, use PBS as the control group, and use an ELISA reader to measure the absorbance at 484nm excitation wavelength, 507nm emission wavelength and OD600. As shown in Figure 2-1-2, the lactate-controlled promoter expressed the strongest fluorescence intensity of eGFP under the condition of 1*10-2M L-lactic acid. This concentration of lactate is similar to the lactate concentration in TME, providing feasibility for application in tumor environment.

The corresponding recombinant plasmid was simulated and synthesized on Snapgene, and the construction scheme and sequence information were sent to GENEWIZ Company for synthesis. The construction scheme is as following:

ALPaGA promoter was used to drive the expression of the downstream gene KYNase. This kind of plasmid contains the KanR resistance gene and a 6xHis tag, which can be used to purify KYNase. In order to efficiently express KYNase, we selected an engineered L-lactate-responsive promoter system (ALPaGA). As a new type of lactic acid-responsive promoter, the researchers combined the LlPRD promoter sequence in wild-type Escherichia coli with the LldR operator sequence to construct a promoter that can work efficiently in lactic acid-rich and oxygen-deficient environments. [30] KYNase is derived from the Pseudomonas fluorescens gene sequence.

1: Marker (Takara); 2-24: PCR; 25: Marker (Takara)

Construction and verification of BL21-pET29a-ALPaGA-KYNase target strain We selected E. coli BL21 (DE3) to make competent cells and transformed the recombinant product pET29a-ALPaGA-KYNase into it. First, take 5μ pET29a-ALPaGA-KYNase plasmid and add 50μ BL21 (DE3) competent cells and mix them evenly. Then, the cells were placed in an ice bath for 30 minutes, heat-shocked at 42°C for 90 seconds, and placed in an ice bath for 2 minutes to transfer the plasmid into the cells. Finally, add an appropriate amount of LB medium (without KanR), mix well, and culture in a shaker for 1 h to allow the cells to recover. Preheat the KanR-resistant solid LB plate medium at 37℃, take an appropriate amount of bacterial solution and evenly spread it on the plate, and invert and culture at 37℃ for 12-16h. As shown in Figure 2-2-3, after the recombinant product is transformed into BL21 (DE3), the bacteria grow normally and are evenly distributed. Pick 1-16 single colonies from the solid plate at random and perform colony PCR identification of the BL21-pET29a-ALPaGA-KYNase target strain. As shown in Figure 2-2-4, the BL21-pET29a-ALPaGA-KYNase colony PCR agarose gel electrophoresis showed that the electrophoresis bands of the 16 single colonies picked were consistent, and the recombinant product pET29a-ALPaGA-KYNase was successfully amplified. The successfully constructed target strain BL21-pET29a-ALPaGA-KYNase was mixed with glycerol and frozen at -80℃.

As shown in Figure 2-2-5, the growth of the target bacteria was consistent with that of the normal bacteria, and the recombinant plasmid had no serious effect on the growth of the bacteria. This provides a theoretical basis for the next KYNase protein expression.

For proteins expressed by inducible plasmids, this experiment set up two large groups, one for L-lactic acid-induced expression of KYNase at 20°C and the other for 37°C. The folding degree of ALPaGA-KYNase protein was roughly evaluated by adjusting the temperature, hoping to obtain better protein expression. After culturing, ultrasonic fragmentation was performed to obtain the induced expression product KYNase, and after SDS-PAGE protein separation, the membrane was transferred, developed, and the image was saved. As shown in Figure 2-3-1, from the development results, it can be seen that the sample band is obvious, the control group has no band, and the background is relatively clear. Compared with the 20℃ induction temperature, the total protein and supernatant bands of the induced group were thicker under the 37℃ induction temperature, and the KYNase protein expression was higher than that of the control group. Combined with the analysis of GAPDPH, the GAPDH internal reference was more uniform under the 20℃ induction condition. At the same time, we also noticed that under the two temperature induction conditions, the GAPDH internal reference had non-specific binding bands, and the primary antibody incubation concentration should be appropriately reduced.

a. Supernatant and precipitated protein samples after KYNase disruption and centrifugationb. Total protein sample after KYNase disruptionc-d. Corresponding GAPDH internal reference protein. MMarker1BL21 (DE3)-pET29a-ALPaGA-KYNase-1-20℃ (L-lactic acid induced) supernatant after disruption2BL21 (DE3)-pET29a-ALPaGA-KYNase-2-20℃ (uninduced) supernatant after disruption3BL21 (DE3)-ALPaGA-pET29a-20℃ crushed supernatant; 4: BL21 (DE3)-20℃ crushed supernatant5BL21 (DE3)-ALPaGA-pET29a-KYNase-1-20℃ (L-lactic acid induced) after fragmentation and precipitation6BL21 (DE3)-ALPaGA-pET29a-KYNase-2-20℃ (uninduced) after fragmentation and precipitation7BL21 (DE3)-ALPaGA-pET29a-20℃ crushed and precipitated; 8: BL21 (DE3)-20℃ crushed and precipitated9BL21 (DE3)-pET29a-ALPaGA-KYNase-1-20℃ (L-lactic acid induced) total protein10BL21 (DE3)-pET29a-ALPaGA-KYNase-2-20℃ (uninduced) total protein after fragmentation11BL21 (DE3)-pET29a-20℃ total protein after disruption; 12: BL21 (DE3)-20℃ total protein after disruption

e. Supernatant and precipitated protein samples after KYNase disruption and centrifugationf. Total protein sample after KYNase disruptiong-h. Corresponding to GAPDH internal reference protein. M: Marker1BL21 (DE3)-pET29a-ALPaGA-KYNase-1-37℃ (uninduced) supernatant after disruption2BL21 (DE3)-pET29a-ALPaGA-KYNase-2-37℃ (L-lactic acid induced) supernatant after disruption3BL21 (DE3)-pET29a-37℃ supernatant after disruption4BL21 (DE3) -37℃ supernatant after disruption5BL21 (DE3)-pET29a-ALPaGA-KYNase-1-37℃ (uninduced) after fragmentation and precipitation6BL21 (DE3)-pET29a-ALPaGA-KYNase-2-37℃ (L-lactic acid induced) after fragmentation and precipitation7BL21 (DE3)-pET29a-37℃ crushed and precipitated8BL21 (DE3) -37℃ fragmentation and precipitation9BL21 (DE3)-pET29a-ALPaGA-KYNase-1-37℃ (uninduced) total protein after fragmentation10BL21 (DE3)-pET29a-ALPaGA-KYNase-2-37℃ (L-lactic acid induced) total protein after disruption11BL21 (DE3)-pET29a-37℃ total protein after disruption12BL21 (DE3) -37℃ total protein after disruption

Select the 20℃ induction temperature group and follow the Western-Blot experimental steps. Use a primary antibody diluent with a dilution concentration of 1:10000 during the primary antibody incubation stage. Figure 2-3-3 shows that when using the primary antibody diluent with adjusted concentration, the GAPDH internal reference band is single and the background is clean.

i. Total protein and supernatant protein samples after KYNase disruption and centrifugationj. KYNase crushed and precipitated samplesk、m. Corresponding to GAPDH internal reference protein MMarker1Total protein after BL21 (DE3) disruption2Total protein after BL21 (DE3)-pET29a fragmentation3BL21 (DE3)-pET29a-ALPaGA-KYNase (uninduced) total protein after fragmentation4Total protein after BL21 (DE3)-pET29a-ALPaGA-KYNase (L-lactic acid induced) disruption5BL21 (DE3) supernatant after disruption6BL21 (DE3)-pET29a supernatant after disruption7BL21 (DE3)-pET29a-ALPaGA-KYNase (uninduced) supernatant after disruption8Supernatant after BL21 (DE3)-pET29a-ALPaGA-KYNase (L-lactic acid induced) disruption9BL21 (DE3) was crushed and then precipitated10BL21 (DE3)-pET29a fragmentation and precipitation11BL21 (DE3)-pET29a-ALPaGA-KYNase (uninduced) fragmentation and precipitation12BL21 (DE3)-pET29a-ALPaGA-KYNase (L-lactic acid induced) fragmentation and precipitation

We first performed protein structure prediction using AlphaFold.

Then, we used FoldX software to mutate each amino acid residue to alanine sequentially and compared the changes in binding energy before and after mutation at each site. The results are shown in Table 3-1-2. The negative value of the modification of canine ureidohydrolase indicates that the binding energy decreases after the mutation, and the protein structure becomes more stable. On the contrary, a positive value indicates that the binding energy increases after the mutation, and the protein structure becomes less stable.

Table 3-1-2 Changes in binding energy at different mutation sites

We have identified several sites that can reduce the binding energy the most. We can then design primers targeting these sites and perform site-directed mutagenesis. We will conduct experiments to determine if this can enhance the enzyme's activity.

First, we amplified the pET-29a(+)-KYNase plasmid by PCR using primers F/R to obtain a linearized fragment. The primers used are listed in Table 3-1-3.

Table 3-1-3 Primers

The amplification procedure is shown in Table 3-1-3.

The PCR-amplified linearized vector (4,606 bp; Fig. 3-1-4a) was treated with DpnI to eliminate residual template DNA. Homologous recombination was then performed, and the product was transformed into DH5α cells (Fig. 3-1-4c), resulting in the successful generation of the mutant plasmid.

(a) PCR amplification results: M: 5000 bp DNA Marker, 1-2: Linearized vector fragments; (b) Plasmid map; (c) Plate map

Individual colonies were selected and grown overnight in LB broth. The plasmids were subsequently purified and subjected to DNA sequencing. As demonstrated in Figure 3-1-5, the sequencing data unequivocally validated the correct assembly of the desired plasmid construct.

To better evaluate the effects of enzyme engineering, we compared the enzymatic activity of the wild-type KYNase with the engineered KYNase. The initial concentration of kynurenine was set at 4.5 mM, and equal amounts of each enzyme were added. The absorbance of the reaction solution was measured every 10 minutes using a microplate reader to assess catalytic efficiency. As shown in Figure 3-2-1, the DA33A mutant exhibited significantly enhanced catalytic efficiency, demonstrating a notable improvement in enzymatic activity compared to the wild-type enzyme.

In order to enable the release of KYNase produced intracellularly after reaching an effective concentration, we designed a cell lysis module based on the lysis protein PhiX174E (91aa). The lysis protein PhiX174E (91aa) is a protein encoded by the phage PhiX174 gene E. It has been shown to be an effective lysing protein in the cellular lysis module. Its mechanism of action is not fully understood, but several studies have shown that the PhiX174E protein triggers cytolysis through membrane binding and oligomerisation of the host cell, as well as a proton-dynamic-dependent step. As shown in Figure 4-1-1, we designed and constructed the plasmid pET29a-ALPaGA-RBS-PhiX174E-T7 and used homologous recombination to integrate the construct. We chemotransformed it into DH5α and extracted the recombinant plasmid after picking a number of single colonies of E. coli on the chemotransfected plates for inoculation.

In order to complete the suicide when the engineered bacteria are released into the environment (natural light), so as to protect the biosafety and avoid genetic contamination, we chose a pDawn-MazF safety module that can initiate the expression of downstream genes under the irradiation of natural light or a single blue light source. pDawn is a blue light-inducible promoter, which can initiate the expression of downstream genes under the irradiation of natural light or a single blue light source expression, and MazF is an RNA enzyme that can target bacterial mRNA for efficient decomposition, thereby inhibiting bacterial growth and reproduction and other life activities.

As shown in Figure 5-1-1, we designed the plasmid pET29a-pDawn-MazF by snapgene software to achieve cleavage and suicide. We successfully constructed the plasmid, and in order to investigate the performance of the population response system, we carried out the measurement of population response growth curve to further prove the feasibility of the project.