Vector construction

Acquisition of Target Fragments

All target fragments in this study were synthesized by Shenzhen BGI Genomics Co., Ltd. after codon optimization. KOD One PCR Master Mix was used to amplify the target fragments from the original plasmids, with the reaction system and procedure shown in Tab. 1 and Tab. 2.

Agarose Gel Electrophoresis and Gel Extraction

Agarose gel powder was weighed according to the required amount, and 1×TAE buffer was added at a ratio of 1% (w/v). The mixture was heated in a microwave oven until the agarose was completely dissolved. When the temperature of the agarose gel solution cooled to approximately 60°C, nucleic acid dye was added. After mixing thoroughly by swirling, the solution was poured into a gel casting tray. A gel comb was inserted, and air bubbles were removed. The gel was left to solidify for approximately 20 minutes.Once solidified, the gel comb was pulled out vertically, and the gel (together with the casting tray) was transferred to an electrophoresis tank. A pipette was used to aspirate the DNA sample, which was then loaded into the gel wells. Electrophoresis was performed at 150 V for 15 minutes. The electrophoresis results were observed and photographed using a gel documentation system.

The target DNA band was excised under an ultraviolet (UV) lamp and transferred to a pre-weighed 1.5 mL centrifuge tube. The tube was weighed again to determine the weight of the gel slice. PE Buffer was added according to the weight of the gel slice. Subsequent gel extraction steps were carried out following the instructions provided with the TIANGEN Agarose Gel DNA Extraction Kit (Enhanced Version). The concentration of the extracted DNA was measured using a spectrophotometer, and the sample was stored in a -20°C refrigerator for later use.

Ligation of the target fragment and the vector backbone

In this study, NEBuilder HiFi DNA Assembly Master Mix was used for all plasmid construction. The reaction system was as follows: when ligating 2 - 3 fragments, the total DNA was 0.03 - 0.2 pmol, and the molar ratio of the insert fragment to the vector was 2:1; when ligating 4 - 6 fragments, the total DNA was 0.2 - 0.5 pmol, and the molar ratio of the insert fragment to the vector was 1:1. 5 μL of 2×HiFi DNA Assembly Master Mix was added, and ddH2O was added to make the total volume up to 10 μL. The reaction program was 50℃ for 30 - 60 minutes.

Transformation of Escherichia coli

The heat shock method was used for transformation, with the following steps:

1. Take out the E. coli DH5α competent cells from the -80°C refrigerator and place them on ice to thaw.
2. Add 5 μL of the recombinant product to 50 μL of DH5α competent cells, and gently tap the tube wall to mix evenly.
3. Incubate on ice for approximately 30 minutes to allow the DNA to attach to the cell surface.
4. Place the tube in a 42°C water bath and heat for 30 seconds to facilitate DNA entry into the cells.
5. After heat shock, immediately transfer the cells back to ice and cool for 2 minutes.
6. Add 900 μL of antibiotic-free LB liquid medium to the centrifuge tube, and incubate at 37°C with shaking at 220 rpm for approximately 1 hour for cell recovery.
7. Spread the recovered cell suspension onto LB agar plates containing kanamycin (Kan) resistance, invert the plates, and incubate at 37°C overnight.

Identification of Escherichia coli Single Colonies

The specific gene fragments were amplified by PCR, and agarose gel electrophoresis was performed to identify whether the target gene was successfully transferred into Escherichia coli cells via the recombinant plasmid. The PCR system and reaction procedure are shown in Tab. 3 and Tab. 4.

Plasmid Extraction from Escherichia coli

Take 50 μL of E. coli bacterial suspension confirmed by PCR identification and add it to 5 mL of LB liquid medium containing Kan resistance. Incubate at 37°C with shaking at 220 rpm for 12-16 hours. Plasmids were extracted following the instructions for the TIANGEN Plasmid Mini-Prep Kit. Subsequently, the extracted plasmids were sent to Tsingke Biotechnology Co., Ltd. for sequencing, and the sequencing results were aligned using SnapGene software.

Construction of Engineering Strain

Vector Linearization

The recombinant plasmid was linearized using the restriction endonuclease NotI-HF. The enzyme digestion reaction system is shown in Tab. 5. The prepared linearization digestion system was incubated in a PCR instrument at 37°C for 2 hours, followed by treatment at 65°C for 20 minutes to inactivate the endonuclease. 1 μL of the reaction product was mixed with Loading Buffer and subjected to agarose gel electrophoresis to verify successful linearization.

Yeast Transformation

The lithium acetate method was used for transformation, with the following steps:

1. Take out the chassis strain from the -80°C refrigerator, spread it on a YPD plate, and incubate at 30°C overnight to activate the strain.
2. Pick the activated strain, spread it on a YPD plate, and incubate at 30°C overnight.
3. In a clean bench, add 90 μL of 50% PEG3350, 5 μL of 2 M lithium acetate, and 5 μL of 2 M DTT to a 1.5 mL centrifuge tube, and vortex to mix.
4. Add 5 μL of denatured salmon sperm DNA to the above centrifuge tube, followed by the linearized vector.
5. Use an inoculating loop to scrape an appropriate amount of bacterial cells, put them into the mixed solution, and gently vortex to disperse the cells.
6. Incubate in a 28°C water bath for 30 minutes, then in a 39°C water bath for 30 minutes, with gentle vortexing every 10 minutes.

Spread the mixture onto the corresponding yeast defective medium plates, invert the plates, and incubate at 30°C for 2 days.

Identification of Yeast Single Colonies

The genomic DNA of oleaginous yeast was extracted using the CTAB method. Using this DNA as a template, we performed PCR to identify whether the target gene had been integrated into the genome of the corresponding colony. The specific steps are as follows:

1. Pick an appropriate amount of bacterial cells and add them to a 50 mL centrifuge tube containing 5 mL of YPD liquid medium. Incubate overnight at 30°C with shaking at 230 rpm.
2. Take 1 mL of the bacterial suspension into a 1.5 mL centrifuge tube, and centrifuge at 12,000 rpm for 5 minutes to collect the bacterial cells.
3. Add 500 μL of CTAB buffer to the bacterial cells, add 2-3 steel beads, vortex for 30 seconds, and repeat this step 3 times.
4. Incubate in a 65°C water bath for 45 minutes, inverting the tube every 15 minutes to mix.
5. In a fume hood, add an equal volume of chloroform to the mixture, invert to mix, and centrifuge at 12,000 rpm for 10 minutes.
6. Pipette 400 μL of the upper layer solution into a new centrifuge tube, add an equal volume of pre-chilled isopropanol, invert to mix, and let stand in a -20°C refrigerator for 30 minutes. Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant.
7. Add 1 mL of 75% ethanol to the precipitate for washing, centrifuge at 12,000 rpm for 3 minutes, and carefully pour off the supernatant. Repeat this step twice.
8. Open the centrifuge tube lid and place it in a 50°C metal bath or oven for 3 minutes to dry the residual liquid at the bottom. A white precipitate can be observed. Add 100 μL of ddH₂O to the bottom of the centrifuge tube, and pipette to dissolve the DNA.

The concentration of the DNA solution was measured using a spectrophotometer. After appropriate dilution, PCR identification was performed. The reaction system and procedure are the same as those in Table 3 and Table 4

Shake Flask Fermentation

Single yeast colonies that had been streaked, activated, and grown well on YPD plates were picked and inoculated into a 24-well deep-well plate containing 1 mL of YPD liquid medium. The culture was incubated at 30°C with shaking at 230 rpm for 16-18 hours.

100 μL of the bacterial suspension was added to a centrifuge tube containing 900 μL of YPD liquid medium for dilution, and the OD600 was measured using a spectrophotometer. Based on the measured OD600, bacterial suspension and YPD medium were added to a 50 mL shake flask to achieve an initial OD600 of 0.1 with a total volume of 10 mL. The culture was incubated at 30°C with shaking at 230 rpm for 3 days.

Extraction and Detection of Lycopene and β-Carotene

Extraction of Lycopene and β-Carotene

Lycopene was extracted from oleaginous yeast via organic solvent extraction, with the specific procedures as follows:

1. Take 100 μL of the fermentation broth into a 1.5 mL brown centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant.
2. Add 900 μL of DMSO (containing 0.01% antioxidant, 2,6-di-tert-butyl-4- methylphenol) to resuspend the cell pellet, and incubate in a 60°C water bath for 1 hour.
3. Add 450 μL of methanol (containing 0.01% antioxidant), and incubate in a 60°C water bath for 30 minutes.
4. Centrifuge at 12,000 rpm for 5 minutes. Pipette 1 mL of the supernatant into a new centrifuge tube, centrifuge again, then transfer 500 μL of the sample into a brown 2 mL screw-cap glass vial. The sample can be directly used for detection and analysis or stored at -20°C under dark conditions.

Qualitative and Quantitative Analysis of Lycopene

Lycopene was qualitatively and quantitatively analyzed at 470 nm using a Varioskan LUX multimode microplate reader or an Agilent 1290 Infinity II liquid chromatography system. For high-performance liquid chromatography (HPLC) analysis, a ZORBAX Eclipse Plus C18 column (4.6 mm × 100 mm; 3.5 μm) was used with the following parameters: injection volume of 5 μL, column temperature maintained at 40°C, mobile phase consisting of acetonitrile:methanol:isopropanol (5:3:2, v/v), and a flow rate of 1 mL/min.

Preparation of the standard curve (Fig. 1 a): The lycopene standard was dissolved and detected using the same method as the samples. The lycopene standard was diluted with a mixed solution of DMSO and methanol (2:1, v/v) to prepare four concentration gradients: 3.125 mg/L, 6.25 mg/L, 12.5 mg/L, and 25 mg/L.

Detection of β-Carotene and Preparation of Standard Curve

Qualitative and quantitative analysis of β-carotene was performed at 450 nm using an Agilent 1290 Infinity II liquid chromatography system. The method was identical to that used for lycopene detection.

Preparation of the standard curve (Fig. 1 b): The β-carotene standard was diluted with a mixed solution of DMSO and methanol (2:1, v/v) to prepare four concentration gradients: 4.6875 mg/L, 9.375 mg/L, 18.75 mg/L, and 37.5 mg/L.

Rational Protein Design to Inactivate Lycopene Cyclase Function in CarRP

Rational Design of CarRP Protein

In this study, REvoDesign was used to predict the monomeric structure of CarRP. Subsequently, the optimal model and the SMILES structure of lycopene were used as input parameters, and DiffDock was employed for blind docking prediction with 100 inference batches and 150 inference steps, yielding complex models and inference trajectories of the two. In PyMOL, all models and trajectories were loaded for alignment analysis, and the optimal theoretical model was jointly determined by referring to the scores and conformational consistency. Based on the selected complex docking model, amino acid residues within 6A of the lycopene molecule were identified as the catalytic pocket, with a total of 27 amino acid sites selected, which are hypothesized to be directly involved in catalysis or substrate binding. Additionally, psi-blast was used for iterative searches against the UniProt90 database to obtain the PSSM (Position-Specific Scoring Matrix). By comparing PSSM data with the spatial structure of the substrate catalytic pocket, 11 catalytic sites with extremely high conservation were identified as targets for function-loss mutation design. Amino acid substitutions with different properties (e.g., hydrophilicity, hydrophobicity, charge) were designed at these sites, aiming to alter the physicochemical properties and functions of the protein while ensuring that these substitutions would not disrupt protein folding, deteriorate thermal stability, or reduce water solubility. Based on the above strategy, 5 substitution mutants were finally selected for subsequent experimental validation

Construction of CarRP Mutant Vectors

Based on the rational protein design structure, mutation primers were designed using SnapGene with the wild-type CarRP sequence as the template. The plasmid pYL-URA2-carRP was used as the template to amplify the fragments and vector backbone. The PCR products were digested with DpnI to remove the methylated plasmid template. Subsequent vector construction was performed according to the method described in vector construction.

The mutant plasmids were linearized and individually transformed into Saccharomyces cerevisiae strain BY4742. Shake flask fermentation was carried out, and the products were detected using HPLC.

High-Density Fermentation of Engineering Strains in Fermenter

Streak the strain on a YPD plate for activation. Pick well-grown yeast single colonies with a deep red color and inoculate them into 5 mL of YPD liquid medium. Incubate at 30°C with shaking at 230 rpm for 16–18 hours. Take 500 μL of the bacterial suspension and add it to 50 mL of YPD medium, then incubate again at 30°C with shaking at 230 rpm for 16–18 hours.

The fermentation broth was inoculated into a 3 L fermenter containing 950 mL of medium for fed-batch fermentation. The fermentation conditions are as follows: 5×YPD was used as the initial medium with the addition of 0.2 M phosphate-buffered saline (PBS). The temperature was maintained at 30°C, the aeration rate was set to 2 vvm, and the agitation speed was controlled between 250–800 rpm to adjust the dissolved oxygen (DO) and maintain it at 20%.

Low-speed feeding was initiated after 48 hours of fermentation. The feed medium composition included yeast extract (100 g/L), peptone (100 g/L), glucose (500 g/L), and 0.2 M PBS. After feeding started, the aeration rate was reduced to 0.3 vvm, and the agitation speed was maintained at 600 rpm. The pH was adjusted to 6.8 by adding 5 M HCl or 5 M NaOH. Samples were taken every 24 hours to determine the OD600 and lycopene yield.

Detection of T5αH-Catalyzed Products

Activate Chassis Strain: Activate the pMASC02-URA-GGPPs-IN-TS chassis strain (currently with the highest taxadiene yield, approximately 6 mg/L) using SD/URA solid deficient medium. Incubate at 30°C with shaking at 220 rpm in an incubator for about 2 days. Activated bacterial cells appear white and plump.For plates (including WT) stored in a 4°C refrigerator for more than 10 days, simply re-streak to activate the strain before starting the small-scale shaking culture.

Activate Cryopreserved Strain: For strains stored in a -80 °C refrigerator, streak them onto a UH plate, incubate at 30 °C for 1 day to allow colonies to grow, and pick 3 single clones.

First Small-Scale Shaking Culture: Use a 24-well deep-well plate (containing 2 mL of SD/UH medium supplemented with 2% glucose). For each strain (including WT), select 3 positive clones, combine them for small-scale shaking culture, and incubate at 30 °C for 1 day. Observe whether the bacterial culture becomes turbid. Incubate together with the pipette tips used for picking colonies (note: this instruction is repeated intentionally).

Second Small-Scale Shaking Culture: Take a new 24-well deep-well plate (containing 3.0 mL of SD/UH medium supplemented with 2% galactose), transfer 1000 μL of the bacterial culture from the first small-scale shaking step, and incubate for 1–2 days.This step aims to standardize the activity of all clones to a similar level, facilitating the subsequent large-scale shaking culture. Note: A wild-type (WT) control (with 3 replicates) must be included in each experiment. Meanwhile, preserve the bacterial strain.

Determine Bacterial Culture Concentration: For the bacterial culture from the second small-scale shaking step, dilute it 10-fold to measure the OD value and record the result.Note: The accurate measurement range of the spectrophotometer is 0.2–0.8, with the highest accuracy between 0.4–0.6. If the value exceeds this range, re-dilute the sample. Therefore, first dilute 1–2 samples 5-fold or 10-fold to determine a reasonable dilution factor before diluting a large number of samples. Before taking each sample, gently pipette the bacterial culture to ensure uniformity.

Large-Scale Shaking Induction: Based on the OD measurement results, transfer the corresponding volume of bacterial culture to a 50 mL Erlenmeyer flask containing 10 mL of medium (SD/UH supplemented with 2% galactose), ensuring the initial concentration of all strains in the large-scale shaking culture is a fixed value (ideally 0.4–0.7; currently adjusted to 0.2–0.3). Incubate at 20 °C with shaking for 5 days. Do not incubate at 30 °C for 1 day, as this will result in no product formation.

Sample Collection and Extraction: Measure the OD value of the bacterial culture and record it (dilute 10-fold or 20-fold based on the approximate concentration, as described above). Transfer the bacterial culture to a 50 mL centrifuge tube, add 5 mL of n-hexane, tighten the cap, and vortex for 5 min (after vortexing, the tube can be temporarily stored in the refrigerator for up to 1 hour). Centrifuge briefly at 3000 rpm, then carefully transfer 4 mL of the upper organic phase to a 15 mL centrifuge tube, preparing for concentration via nitrogen blowing (using compressed air with air filtration). First, take a small sample for preliminary on-machine testing; then concentrate the remaining sample.Notes: Do not aspirate the lower aqueous phase or suspended particles; use tubes with good airtightness.Note: n-hexane is toxic and highly volatile; operate in a fume hood.

Concentrate Product: Place the 15 mL centrifuge tube on a nitrogen evaporator for concentration. When the volume is reduced to 1.5 mL, remove the tube, tighten the cap, and shake thoroughly to dissolve any residual product on the tube wall with n-hexane. Centrifuge briefly at 3000 rpm, then transfer the solution to an imported 1.5 mL centrifuge tube for secondary concentration until no solution remains.Notes: The product is volatile; do not extend the concentration time excessively. Stay beside the tube during the second concentration and stop immediately once the solution is dried, then seal the tube.

Solvent Preparation (taking dodecane as an example; the same applies to n-hexane):Prepare dodecane containing 2 mg/L β - caryophyllene: Configure an appropriate volume as needed. For example, to prepare 1 mL of solvent: mix 990 μL of dodecane with 10 μL of 200 mg/L β-caryophyllene.When dissolving samples for on-machine testing (whether using n-hexane or dodecane), add β-caryophyllene to a final concentration of 2 mg/L.

Determine Product: Take 100 μL of solvent containing 2 mg/L β-caryophyllene, add it to the 1.5 mL centrifuge tube with the dried sample, and vortex thoroughly to dissolve substances remaining on the tube wall. Centrifuge at 12000 rpm at low temperature (10 °C) for 30 min, then transfer 90 μL of the supernatant to the liner of a sample vial (avoid aspirating the lower precipitate as much as possible) and seal the vial tightly with a non-pre-slit cap. Proceed with on-machine detection.Note: n-hexane is extremely volatile; ensure the vial cap has good airtightness. Store the prepared sample in the refrigerator after preparation.

Statistical Analysis

Each experiment was repeated at least 3 times, and the results are expressed as the mean value. Statistical analysis of the experimental results was performed using GraphPad Prism 9. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), and comparisons between two groups were performed using the independent samples t-test. A P-value less than 0.05 was considered statistically significant.

Media

1. Glycerol cryo-stocks

Add 250 μL 80% glycerol to 750 μL liquid culture, store at -20°C

2. Lysogeny Broth

For L liter:

For selective medium: Add 100 μL of 100 ng/μL stock of ampicillin to 100 mL of LB for plates.

3. YPD

For L liter

For plates: Add 2% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if needed, pour plates (20 ml/plate needed)

4. 2% CTAB

For L liter

5. Yeast Complete Medium

For L liter