Results

Gene cloning

1.1 crnA

We revived Pseudomonas putida NBRC 14164 from our laboratory's bacterial culture collection and extracted its complete genome using the TIANamp Bacteria DNA Kit (Cat.#DP302-02). Primers were designed with KpnI and BamHI restriction enzyme sites (as shown in Figure 1) and amplified by PCR (all primers, and formulations are provided in the supporting information).

1.2 creJ

Since Arthrobacter nicotianae strain 23710 is not currently preserved in our laboratory, we ordered the gene sequence from BGI Genomics. We amplified the sequence from the PMV plasmid with the fused creJ gene obtained from the company. This sequence will be used for homologous recombination with the pcs27 plasmid.

The amplification results are shown in Figure 2.

Co-expression plasmid construction

As part of the module validation, we wanted to investigate the performance data of the creatinine degradation module. Therefore, we conducted separate fermentation and supplementation experiments for this module. Considering the requirements for the expression levels of these two enzymes, we selected the pCS27 plasmid with medium copy number and used the lac promoter to express the desired proteins under IPTG induction.

To do this, we separately constructed two plasmids, pCS-lac-crnA and pCS-lac-creJ, with the primers as mentioned above. Additionally, we amplified the plasmid backbone of pCS27 and the homologous recombination fragment of the glutamate racemase (details can be found in the complete pathway fermentation experiment). We first connected the three fragments using overlap PCR and then performed homologous recombination with the plasmid backbone to obtain the required plasmid for this module.

The plasmid map is shown in the figure.

Fermentation Additive Experiment

3.1Amplification and Validation of Plasmids

We will amplify the constructed plasmid in E.coli DH5α through chemical transformation method. The transformed cells will be spread onto solid LB agar plates supplemented with kanamycin for antibiotic selection. Positive colonies will be selected (Figure 4) and subjected to colony PCR screening (Figure 5) using primers overlapping the target region. The colonies carrying the correct plasmid will be cultured in liquid LB medium supplemented with kanamycin at 37°C on a shaker overnight. Plasmid DNA will be subsequently extracted. To ensure the correctness of the plasmid sequence, it will be sent to a genetic company for sequencing, confirming its accuracy.

3.2Additional experiments

Experimental Method: The carrier bacterium to be used, Nissle 1917 (EcN), was pre-cultured overnight at 37°C in LB medium without antibiotics. Plasmid transformation was carried out using electroporation, and the transformed plasmids were selected on solid LB medium with kanamycin resistance. Positive colonies were picked and inoculated into liquid LB medium with kanamycin resistance, followed by overnight incubation at 37°C. Subsequently, they were inoculated into 50 mL of M9 medium with kanamycin resistance, induced with IPTG, and supplemented with the substrate creatinine (1 mM). The cultures were then incubated on a shaker at 30°C for 48 hours.

At 12, 24, 36, and 48 hours of fermentation, samples were taken from the fermentation broth, processed, and subjected to High-Performance Liquid Chromatography (HPLC) analysis to obtain the fermentation data for this module.

Compared with the blank control, our results show that the strain after our transformation can metabolize creatinine and transform it well, which symbolizes that the construction of our first module is successful, which is a very gratifying result. At the same time, through the investigation of the literature, we found that our results were close to the existing literature reports.Compared with the blank control, our results show that the strain after our transformation can metabolize creatinine and transform it well, which symbolizes that the construction of our first module is successful, which is a very gratifying result. At the same time, through the investigation of the literature, we found that our results were close to the existing literature reports.

Gene cloning

1.1 UreABC

As part of the structural protein of urease, the gene cluster of urease has been widely reported. We used the urease gene cluster from Providencia rettgeri strain JNB815. Since this strain was not preserved in our laboratory, we purchased it from the China Center of Industrial Culture Collection (CICC).

Similarly, we revived this strain and extracted its genome using the same method. We amplified this sequence through PCR. Considering expression levels, we used our laboratory's high-copy plasmid, pETlac, as the vector and performed homologous recombination with the gene clone.

1.2 ureEFGD

As an important auxiliary structure of urease, we also obtained it from the genome of Providencia rettgeri strain JNB815 and recombined it into the pETlac plasmid using the same method.

Co-expression plasmid construction

Until now, there have been very few reports on the soluble expression of urease in Providencia rettgeri strain JNB815. Using pETlac-ureABC and pETlac-ureEFGD as templates, we amplified the two expression frames separately. After connecting them, we homologously recombined them with the pETlac plasmid backbone to obtain this plasmid."

Fermentation Addition Experiment

3.1 Plasmid Amplification and Verification

We will amplify the constructed plasmid by chemical transformation in E. coli DH5α and then spread it on ampicillin-resistant solid LB agar plates for resistance selection. Positive colonies will be selected (Figure 15) and subjected to colony PCR screening (Figure 5, using ureABC as PCR primers) to obtain strains carrying the correct plasmid. These strains will be cultured in liquid LB medium supplemented with ampicillin resistance at 37°C with shaking overnight. Subsequently, plasmids will be extracted to ensure the correctness of the plasmid sequence. They will be sent to a gene company for sequencing to confirm their accuracy.

3.2 Soluble Expression of Urease

In E. coli expression systems, inclusion bodies can form when expressing foreign proteins due to the lack of modification systems. Currently, the mainstream approach in the scientific community to increase solubility is to purify and refold the expressed proteins or use molecular chaperones or solubilization tags. Therefore, BUCT has attempted four molecular chaperones to enhance solubility: GroEL/GroES, DnaK/DnaJ, IbpAB, and sigma32. The plasmid maps for each of these chaperones are shown in the figure below.

Solubility Verification:

The gene segments constructed as described above were subjected to homologous recombination with the pET-duet plasmid using the same method. E. coli BL21 cells, which had been pre-cultured and stored, were revived. The plasmids were transformed into the host cells through electroporation. Four different molecular chaperones were separately introduced into the host cells, and a control group with an empty vector and another control group with no molecular chaperones were included, resulting in a total of six groups.After selection for antibiotic resistance, the cells were initially inoculated into LB medium containing both ampicillin and kanamycin antibiotics. Subsequently, they were transferred to 50 ml of LB medium containing both antibiotics in shake flasks and cultured at 37°C until the OD reached the desired range (0.2-0.6). The cultures were induced with 0.5 mM IPTG and further incubated for 12-16 hours.The fermentation broth was then collected and subjected to repeated sonication for cell disruption. Each group was divided into two fractions: the supernatant and the precipitate. Finally, the gel electrophoresis results for this protein were obtained using SDS-PAGE.

After comparison, it is easy to notice that the effect of GroEL/GroES is the most excellent.

3.4 Additional experiments

Experimental Method:

The carrier strain, Nissle 1917 (EcN), to be used in the experiment is pre-cultured overnight at 37°C in LB medium without antibiotics. The co-expression plasmid and chaperone plasmid are transformed into the carrier strain using electroporation. The transformed cells are then screened on solid LB medium containing both ampicillin and kanamycin as dual resistance markers. Positive colonies are selected and inoculated in liquid LB medium containing ampicillin and kanamycin at 37°C overnight. The cells are subsequently inoculated into 50 ml of M9 medium containing ampicillin and kanamycin, induced with IPTG (Isopropyl β-D-1-thiogalactopyranoside), and supplemented with urea (10g/L). The cultures are incubated in a shaker at 30°C for 48 hours.

Samples of the fermentation broth are taken at 12, 24, 36, and 48 hours of fermentation. The samples are processed and analyzed using High-Performance Liquid Chromatography (HPLC) to obtain fermentation data for this module. Due to the inherent metabolic activities of Escherichia coli, it is difficult to measure the exact amount of ammonia produced. However, fortunately, E. coli itself cannot directly metabolize urea. Therefore, the decrease in urea concentration can directly reflect the performance of this module.

In fact, although we have made a lot of efforts, including literature research and group discussion, we still haven't got an absolutely effective solution to the problem of urease soluble expression, which is undoubtedly regrettable, but we also know that the process of scientific research is full of challenges, so we will continue to explore this problem and strive to solve this problem!

Gene cloning and plasmid construction

1.1 racE

Because the synthesis of polyglutamic acid requires equal amounts of L-glutamic acid and D-glutamic acid, the addition of glutamate racemase can increase the yield of polyglutamic acid. Therefore, we identified the glutamate racemase gene racE in Bacillus amyloliquefaciens LL3 and directly ordered it from a genetic company. We amplified the gene fragment for homologous recombination directly from the obtained PMV plasmid containing this gene. This gene fragment was then homologously recombined with the pcs27 plasmid backbone to obtain pcs-lac-racE.

1.2 pgsBCAE

The pgs promoter from Bacillus subtilis strain A-5 plays a crucial role in the synthesis of polyglutamic acid. It utilizes racemic glutamic acid as a raw material to synthesize polyglutamic acid and then expels the product extracellularly. We obtained it through PCR from the genome of Bacillus subtilis strain A-5 and recombined it with the pETlac plasmid backbone.

ygay-50trc-gdhA gene integration

Because this module requires glutamic acid as a precursor, and in the metabolic system of E. coli, the gdh pathway is one of the major pathways for glutamic acid production. Therefore, we identified the gene encoding glutamate dehydrogenase, gdhA, in E. coli. We replaced the constitutive promoter -50trc. Additionally, to reduce the burden on the bacterial cells, we used CRISPR-Cas9 technology to target the ygaY locus in the genome for integration. After integration was completed, we induced the elimination of the PT plasmid using L-arabinose and cultured the cells at 42°C for 12 hours to eliminate the pCas9 plasmid.

Fermentation Additions Experiment

Because the gene-integrated vector strain is still under preparation prior to this fermentation experiment, the wild strain is still being used for fermentation.

Method:

The carrier strain to be used, Nissle 1917 (EcN), was pre-cultured overnight at 37°C in LB medium without antibiotics. Both plasmids mentioned above were co-transformed using electroporation, and positive colonies were selected on solid LB medium with double resistance to ampicillin and kanamycin. The selected colonies were then inoculated into liquid LB medium with double resistance to ampicillin and kanamycin and cultured overnight at 37°C. Subsequently, they were inoculated into 50 ml of M9 medium with double resistance to ampicillin and kanamycin, induced with IPTG, and supplemented with the substrate glutamic acid (5g/L). The cultures were shaken at 30°C for 48 hours.

At the 12th, 24th, 36th, and 48th hours of fermentation, samples of the fermentation broth were taken respectively. After processing the samples, they were subjected to High-Performance Liquid Chromatography (HPLC) analysis to obtain fermentation data for this module.

The experimental results show that our construction of the pathway of γ-PGA is quite successful. The modified E. coli can effectively convert glutamate into γ-PGA. Although the yield cannot reach the highest yield in the world at present, this may be related to the use of different carrier bacteria, but this result still shows that our construction of polyglutamic acid synthesis module is successful.

Plasmid constrution

In order to construct a complete metabolic pathway, we will create two co-expression plasmids.

1.1 pCS27-lac-crnA-creJ-racE

Considering the requirements for expression levels, excessive expression in the front part of the pathway can lead to the accumulation of intermediates such as ammonia, which has a negative impact on the strain. In addition, based on reports regarding the activity of various enzymes, we have made certain adjustments to the gene positions.

The construction method of this plasmid has been described in detail in the creatinine degradation module.

1.2 pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCA

After conducting experiments in the urea degradation module, we found that even with the assistance of molecular chaperones, the activity of urease was relatively weak compared to other enzymes in the pathway. Recognizing the significance of accessory proteins in enhancing its enzymatic activity, we decided to replace the constitutive promoter with a higher strength one in an attempt to increase urease activity. To do this, we first used the pZe12 plasmid for homologous recombination to obtain pZe-plpp1.2-ureEFGD. Then, we amplified the entire expression cassette through PCR and, using the same method, recombined the resulting lac-ureABC and lac-pgsBCAE with the pETlac plasmid backbone after overlap connection.

Additional experiment

After successfully constructing the plasmids and confirming their integrity through company sequencing, our operational procedure was similar to the previous one, with the exception that we used an engineered integrative strain as the host. The host strain we used was Nissle 1917 (EcN), which had been integrated with ygay-50trc-gdhA. This strain was pre-cultured overnight at 37°C in LB medium without antibiotics. Subsequently, we performed co-transformation of the two plasmids and the molecular chaperone using electroporation. Selection was carried out on solid LB medium containing both ampicillin and kanamycin, and positive colonies were picked and inoculated into liquid LB medium containing both antibiotics. The culture was incubated at 37°C overnight.

The next step involved inoculating the strain into 50 mL of M9 medium without a nitrogen source but with dual resistance to ampicillin and kanamycin. After induction with IPTG, we added substrates creatinine (1 mM) and urea (10 g/L) and cultured the cells on a shaker at 30°C for 48 hours.

At 12, 24, 36, and 48 hours of fermentation, samples were taken from the culture. These samples were processed and subjected to High-Performance Liquid Chromatography (HPLC) analysis, yielding fermentation data for this module.

The construction of the complete pathway and the results of the fermentation experiment are quite exciting, as it indicates that we have successfully constructed this pathway. Although our production is relatively low, this may be due to the poor actual effect of urease, it still marks a milestone step for BUCT in solving the pain of CKD patients!