• Preparation of BW/pKD46 Electrocompetent Cells:
• Introduced the up-kan-down targeting fragment into the prepared competent cells.
• Performed colony PCR verification; the expected target band was 600 bp, but the results were incorrect.

Replacement of Primers and Colony PCR:

• New primers were designed and used to perform another round of colony PCR to improve the accuracy of the guaB gene knockout verification.

Selection of Single Clones for Validation:

• Three single colonies (No. 6, No. 10, and No. 14) were chosen for further validation of the guaB gene knockout.

Validation on Selective Media:

• The selected clones were cultured on M9 minimal medium plates, with or without the addition of 0.2 mM xanthine, to assess their growth.

Incubation:

• The cultures were incubated in a shaking incubator at 37°C for 24 h to monitor the growth patterns and confirm the gene knockout.

The growth results on the selective media will help us determine if the guaB gene has been successfully knocked out in the selected clones.

Verification by Streaking Method:

• The selected E. coli BW25113 clones were streaked onto agar plates to validate the knockout of the guaB gene.

Observation of Growth Results:

• The results confirmed successful gene knockout. The clones were able to survive on the medium containing xanthine, but showed no growth on the medium without xanthine.

These data indicated that the guaB gene knockout was successful, as the knockout strains exhibited the expected growth dependency on xanthine

Streaking on Kanamycin-Resistant Medium:

• The verified guaB knockout strains were streaked onto agar plates containing kanamycin to assess their resistance and confirm the presence of the knockout cassette.

Selection and Spotting of Single Clones:

• Single colonies were picked and first inoculated on a plate with A+K (ampicillin and kanamycin) resistant medium, followed by inoculation on a plate with K (kanamycin only) resistant mediumto evaluate their growth patterns.

Identification of Correct Knockout Strains:

• Strains that could grow on kanamycin (K) plates but failed to grow on A+K plates were selected for further propagation. These growth patterns indicate the successful knockout of the guaB gene and the appropriate loss of the pKD46 plasmid (ampicillin resistance).

This procedure helps us confirmthat the strains are correctly knocked out for the guaB gene and do not carry any unwanted plasmid, which is critical for subsequent experimental steps.

Preparation of Competent Cells:

• The verified guaB knockout strain (BW25113 ΔguaB) was prepared as competent cells for further transformation.

Transformation with pYB1s-ndmDBACE Plasmid:

• The plasmid pYB1s-ndmDBACE was introduced into the competent BW25113 ΔguaB strain.
• After transformation, the cells were incubated in a 37°C shakerfor 16 h. However, the transformed strains could poorly grow.

Revival and Re-plating:

• The BW25113 ΔguaB strain was revived and spread onto K30+S (kanamycin and spectinomycin) resistant agar plates to promote better growth conditions.

Re-transformation with pYB1s-ndmDCBAE Plasmid:

• The pYB1s-ndmDCBAE plasmid was reintroduced into the BW25113 ΔguaB strain to ensure successful transformation.

These steps aim to optimize the transformation conditions and confirm the stable integration of the desired plasmid into the guaB knockout strain for subsequent experiments.

Comparison of Growth Curves:

• Conducted a comparative analysis of the growth curves between the wild-type E. coli BW25113 strain and the guaB knockout strain (BW25113 ΔguaB).

Observation and Analysis:

• The growth patterns were monitored under identical conditions to evaluate the impact of the guaB gene knockout on bacterial growth.
• Differences in growth rates, lag phases, and overall cell density were carefully recorded to assess the physiological effects of the gene knockout.

This comparison provides crucial insights into the metabolic and growth characteristics of the guaB knockout strain, confirming the gene knockout's effects and helping to guide further optimization in the engineered strain's use for downstream applications.

Targeted Site-Directed Mutagenesis:

• Specific pocket residues within the ndmB enzyme were selected for site-directed mutagenesis to potentially enhance the enzyme's catalytic activity.
• Mutations were introduced at these sites to explore their impact on the enzyme's structure and function.

Detection Using Enzyme Assays:

• The activity of the mutated ndmB variants was assessed using a plate reader.
• This initial screening aimed to identify any changes in enzymatic activity due to the introduced mutations.

HPLC Analysis:

• High-Performance Liquid Chromatography (HPLC) was employed to detect the production of 7-MX or other relevant metabolites.
• Unfortunately, the HPLC analysis showed no detectable product formation, suggesting that the mutations did not enhance the enzyme's activity.

Optimization of PCR Conditions:

• To improve the mutagenesis efficiency, PCR conditions were adjusted, specifically modifying the concentrations of Mn2+ and dNTPs:

0.01 mM Mn2+ with dNTPs

0.02 mM Mn2+ without dNTPs

0.01 mM Mn2+ without dNTPs

These steps aimed to generate ndmB mutants with enhanced functionality, but further optimization and additional mutagenesis strategies may be required to achieve the desired increase in enzyme activity and product yield.

• Use gel extraction to recover the vector and mutated fragments.
• Perform Gibson assembly (C116).
• Transform the competent cells, extract plasmids, send plasmids for DNA sequencing, and check the mutation rate.

Based on the results, we found that mutations only occurred in the SPRBCC region under the condition of 0.02 mM Mn2+.

Therefore, subsequent experiments will continue to use 0.02 mM Mn2+.

PCR with 0.02 mM Mn2+ and dNTPs:

PCR with 0.02 mM Mn2+ without dNTPs:

• Gel extraction was used to recover the vector and mutated fragments.
• Gibson assembly (C116) was performed.
• Transformed cells were cultured, plasmids extracted, sent for sequencing, and mutation rates were checked.

There were many cloned with unmutated sequences.

Site-Directed Mutagenesis of ndmB:

• Site-directed mutagenesis was performed at potential pocket sites on ndmB (2012).
• Degenerate primers were used for PCR on the plasmid (pYB1s-ndmDtB) to induce mutations at Trp256, Cys267, and Met27.
• The product was purified using DpnI digestion and transformed into BW25113 for functional validation.

Verification of Random Mutagenesis Conditions for ndmB:

• Gel extraction was used to recover the vector and mutated fragments.
• Gibson assembly (C116) was performed.
• Single colonies were picked, plasmids were extracted followed by DNA sequencing, and mutation rates were evaluated.

Construction of pYB1s-ndmDCE Plasmid:

• The pYB1s-ndmDCE plasmid was constructed and transformed into BW25113-△guaB.
• If caffeine degradation is efficient, the produced PX will be converted into xanthine, allowing E. coli to survive.
• If caffeine degradation is inefficient, a large amount of caffeine will be converted into 3-MX, which does not support E. coli surviving.

Colony PCR results (using ndmA-F and ndmA-R primers):

Functional Validation of pYB1s-ndmDCE Plasmid:

• The mutated plasmid pYB1s-ndmDtBs was obtained.
• Expression was induced in deep-well plates.
• Supernatant from the transformed bacteria was transferred to a 96-well plate, and BW-△guaB+DCE was added.
• Growth curves were generated .

6.6-6.13 — Functional Validation of pYB1s-ndmDCE

• The correctly sequenced pYB1s-ndmDCE plasmid was transformed into BW△guaB, and single colonies were picked for induction.
• ZY medium containing xanthine was removed and the bacteria were washed; the supernatant was discarded after centrifugation, and cells were washed once with M9 medium. The cells were then resuspended in 5 mL of M9 medium.

6.14-6.19 — Feasibility Testing of Current Conditions for Screening ndmBs

• Verified whether induction at 25°C in deep-well plates (ZYM-5052) and whole-cell catalysis in M9 medium were feasible.
• pYB1s-ndmDtA plasmid was transformed into BW25113 as a high-efficiency control group.
• pYB1s-ndmDtB plasmid was transformed into BW25113 as an experimental group.
• pYB1s-ndmDtA showed nearly 100% conversion of TP to 3-MX, proving the feasibility of the method.
• pYB1s-ndmDtB showed an average conversion rate of 6.9% from TP to 1-MX.

Comparison of pYB1s-ndmDtA and pYB1s-ndmDtB.

• Induced pYB1s-ndmDCE (BW25113) by adding a small amount of xanthine powder.
• After induction, washed cells with M9 medium and inoculated 10% of the bacteria into M9 medium containing 7-MX.
• Incubation for 36 h to observe difference between media with and without 7-MX.
• Growth inhibition or cell death only began after initial growth (possibly due to incomplete dissolution of 7-MX during induction); without 7-MX, growth was very slow.
• LB medium's yeast extract also contains some intermediate metabolites like xanthine; different amounts of yeast extract were added, and growth curves were plotted in a 96-well plate.

• The results showed that the xanthine content in the LB medium was insufficient, and doubling the yeast extract could better supplement it.
• Subsequently, the “LB Y2” medium (with double the yeast extract of LB) was used to cultivate and induce the strains.

• pYB1s-NdmDtBA can degrade caffeine into 7-MX.
• pYB1s-ndmDCE can further degrade 7-MX into xanthine.
• Used pYB1s-NdmDtBA as the production strain and pYB1s-ndmDCE as the testing strain.

Testing of pYB1s-ndmDCE:

• Introduced different substrates (compounds in the xanthine family ) to the BW25113 strain with the guaB gene knocked out, and examine bacterial growth status.
• The growth status of the strain was poor, indicating that this testing method was not effective to be used for the specificity verification of 7-MX.

• pYB1s-NdmDtBA can degrade caffeine into 7-MX.
• pYB1s-ndmDCE can degrade 7-MX into xanthine.
• Used pYB1s-NdmDtBA as the production strain and pYB1s-ndmDCE as the testing strain.
• The first round of testing was completed.
• The first round of testing showed poor growth status of the strain when measuring OD values using a microplate reader.

• Analyzed the second round of screening results and identified an advantageous strain, 4-C5.

• Conducted sequence comparisons between the two rounds of results, identifying position 289 as the most crucial residue .

Used pYB1s-NdmDtBA as the production strain and pYB1s-ndmDCE as the test strain, conducting three rounds of screening (only targeting mutations at position 289).

• The mutated plasmid was first transformed into DH5α, with single colonies grown and washed in water; plasmids were extracted and then transformed into BW25113, spread on square plates.
• Single colonies were picked and inoculated into deep-well plates (800 μL of ZY + arabinose + IPTG + S50), with the first four wells containing non-mutated strains, and then induced the bacteria at 25°C for 18 h.
• Whole-cell catalysis was conducted at 25°C for 18 h.
• After centrifugation, 200 μL of supernatant was added to a 96-well plate with 10 μL of a supplement solution (containing glucose, calcium chloride, magnesium chloride, trace elements, inducers, and antibiotics).
• An additional 10 μL of the induced DCE was added.

  (Due to the apparent slow growth of DCEA, delayed induction may be required; the 96-well plate was sealed in a plastic bag and stored at 4°C until DCE induction was complete.)

• Growth was measured using a microplate reader, with successful growth indicating the ability to produce 7-MX.

Analysis of the second-round screening results:

• Selected some samples for sequencing.
• The relationship between amino acids and yield after mutation was analyzed by GPT.

• Tested different Anderson strong promoters for ndmBs.
• After construction, liquid-phase testing revealed the presence of by-products during 7-MX production.

Screening of ndmBs Mutants:

• After plasmid transformation into strains, single colonies were separated by streaking on plates, and the plasmid was extracted and digested with KpnI and XbaI. The correct bands were identified at 2448 and 3272, which were verified by DNA sequencing analysis.

1. pYB1s-ndmDtBs X289 was transformed into BW25113.

2. pYB1s-ndmDtBs X289 with CRS and pSB1c-frmAB-FDH were co-transformed into BW25113.

Induction at 25°C for 18 h:

Single transformation results:

1-G2 (CCC-289): Pro=P289 - No bacterial growth.

2-G2 (TCA-289): Ser=S289 - Cell lysis upon induction.

1-B12 (GCA-289): Ala=1A289 - OD=13.7, white color, foul odor, apparently contaminated .

2-B12 (GCA-289): Ala=2A289 - Normal OD=7.45.

Protein analysis planned.

• Induction and whole-cell catalysis were performed on 1-G2, 2-G2, 1-B12, and 2-B12 strains, and samples were prepared for HPLC.
• SDS-PAGE was conducted.

• Repeated whole-cell catalysis for 1-G2 and 2-G2 strains (unfortunately contaminated).

• Whole-cell catalysis (20°C, 20 h): Fermentation broth was freshly prepared (caffeine + Tris-HCl pH 9) and operated on ice.
• OD gradient (8 mM): 30, 50, 70, 100.
• Substrate gradient (50 OD): 1, 2, 4, 8, 10.
• Samples were prepared for testing under the best conditions.

1. Induction of B12 ndmBs + CRS dual-transformation strain is in progress.

2. Transformation and plating of the new C12 (289Ala) strain is underway.

• Induction: Cultivated at 37°C for 1.5 h before adding the inducer until OD was between 0.2 and 0.6.
• Transferred to 25°C, induced for a total of 18 h, and the final OD was approximately 7-8.
• Whole-Cell Catalysis: Whole-cell catalysis (20°C, 20 h) involved transferring the cell suspension (10 mL centrifuge tube with 1 mL fermentation broth) to a 100 mL Erlenmeyer flask and shaking.
• OD gradient (10 mM): 10, 20, 40, 80, 100.
• Substrate gradient (50 OD): 1, 2, 4, 8, 10.
• Sample Preparation: Directly added Tris-HCl (pH 9) to the whole-cell catalysis completion flasks, diluted to a substrate concentration of 2 mM, and added 4 mL Tris-HCl to the 10 mM substrate group, reaching a total volume of 5 mL. Similarly, diluted the 4 mM substrate to a total volume of 2 mL.
• Samples were mixed, 1 mL was transferred to an Eppendorf tube, centrifuged at 10,000 g for 10 min, and the supernatant was passed through a 0.22 μm filter membrane into HPLC vials.
• The yieldsand time curve were obtained.

1. Induction of B12 ndmBs + CRS dual-transformation strain is in progress.

• Focused on optimizing the production of the B12 strain based on the first round of fermentation conditions.
• Re-induced and fermented:

Induction: Cultivated at 37°C for 1.5 h before adding the inducer until OD was between 0.2 and 0.6.
Transferred to 25°C, starting from the time of transferring at 37°C, induced for a total of 18 h, and the final OD was approximately 7-8.

• Whole-Cell Catalysis: Whole-cell catalysis (20°C, 20 h) involved transferring the cell suspension (10 mL centrifuge tube with 1 mL fermentation broth) to a 100 mL Erlenmeyer flask and shaking.
• OD gradient (10 mM): 10, 20, 40, 80, 100.
• Substrate gradient (50 OD): 1, 2, 4, 8, 10.

• Sample Preparation: Directly added Tris-HCl (pH 9) to the whole-cell catalysis completion flasks, diluted to a substrate concentration of 2 mM, and added 4 mL Tris-HCl to the 10 mM substrate group, reaching a total volume of 5 mL. Similarly, diluted the 4 mM substrate to a total volume of 2 mL.
• Samples were mixed, 1 mL was transferred to an Eppendorf tube, centrifuged at 10,000 g for 10 minutes, and the supernatant was passed through a 0.22 μm filter membrane into HPLC vials.

• Collected coffee grounds from various stores, including Starbucks, Luckin, Kudi, 818, KFC, and Shengji.

• Dried the grounds for a day but found that they were still moist.
• Measured 10 g of coffee grounds from each brand, placed them in Erlenmeyer flasks, and dried them.
• Added water at four times the mass-to-volume ratio to the dry coffee grounds from each brand to extract caffeine.
• Placed the flasks in an ultrasonic cleaner (15 W) for 10 min to facilitate caffeine dissolution.
• Collected 1 mL of the liquid from each sample and centrifuged at 15,000 g for 3 min.
• Prepared samples and performed HPLC analysis.

• Collected coffee grounds from various stores, including Starbucks, Luckin, Kudi, 818, KFC, and Shengji.
• Diluted dry coffee grounds at a mass-to-volume ratio of 4:1 to prepare transformation solutions.

• Returned to stores to collect more coffee grounds.
• Added water at a mass-to-volume ratio of 4:1 to the dry coffee grounds from each brand to extract caffeine.
• Placed the flasks in an ultrasonic cleaner (15 W) for 10 min to facilitate caffeine dissolution.
• Collected 1 mL of the liquid from each sample and centrifuged at 15,000 g for 3 min.
• Prepared samples and performed HPLC analysis.

• Used B12 dual transformation strain.
• Added water at a mass-to-volume ratio of 4:1 to the dry coffee grounds from each brand to extract caffeine.
• Placed the flasks in an ultrasonic cleaner (15 W) for 10 minto facilitate caffeine dissolution.
• Collected 1 mL of the liquid from each sample, centrifuged at 15,000 g for 3 min, and prepared samples (due to high levels of impurities, filtered twice through a membrane).
• Prepared samples with 5 g each of Luckin and Starbucks coffee grounds, dissolved in 20 mL, 40 mL, 80 mL, and 100 mL of water respectively, and sonicated for 10 min.
• Collected 1 mL, centrifuged at 15,000 g for 5 min, collected the supernatant, centrifuged again, and filtered twice through a 0.22μm membrane.
• Placed the flasks in an ultrasonic cleaner (15 W) for 10 minto facilitate caffeine dissolution.
• Collected 1 mL of the liquid from each sample, centrifuged at 15,000 g for 10 min, and prepared samples.

• Weighed 5 g of coffee grounds from each brand and dissolved them in 100 mL of water.
• Heated to boiling point five times using a microwave.
• Filtered using filter paper, collected 1 mL of the filtered liquid, and centrifuged at 15,000 g for 5 min.
• Used a 1 mL syringe to collect the centrifuged liquid and filtered it twice through a membrane.

• Weighed 2 g of coffee grounds from each brand and dissolved them in 5 mL of Tris buffer.
• Heated to boiling point five times using a microwave.
• Filtered using filter paper, collected 1 mL of the filtered liquid.
• Centrifuged at 15,000 g for 5 min, used a 1 mL syringe to collect the centrifuged liquid, and filtered it twice through a membrane.
• From 0 h, samples were collected every 2 hfor a total period of 18 h.
• For each sampling, 50 μL of the transformation liquid was mixed with 50 μL of Tris buffer, centrifuged at 15,000 g for 10 min, and then prepared for HPLC analysis.
• Performed HPLC to produce time curves for 7-MX production from different brands of coffee grounds.