Sequence Design and Optimization

To construct engineered strains capable of synthesizing neo hesperidin and hesperetin, we selected key biosynthesis genes and performed codon optimization for E. coli. All coding sequences were synthesized by GenScript, eliminating EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI restriction enzyme sites while maintaining function, and conforming to RFC#10 standards:

• ThF3′H (from Tricyrtis hirta), CPR (from Arabidopsis thaliana), and MpOMT S142V (from Mentha × piperita), for hesperetin synthesis;
• UGT73B2, VvRHM-NRS, and Cm1,2RhaT, for neo hesperidin synthesis.

Additionally, regulatory elements were designed and constructed:

• pCadC (acid-responsive promoter, GenScript) for pH-inducible expression;
• pBAD (arabinose-inducible promoter, BBa_K808000);
• pCspA (cold-shock inducible promoter, BBa_K3289001).

All constructs were verified by sequencing (Eurofins Genomics, USA).

Genetic Construct Validation

PCR PCR Reaction Mixture (50 μL) All PCR reactions used high-fidelity DNA polymerase to ensure amplification accuracy. The reaction mixture was prepared as follows (using nuclease-free, DEPC-treated water):

Component	Volume
dNTP Mix (2.5 mM each)	4 μL
10× Pfu Buffer	5 μL
Pfu DNA Polymerase	0.5 μL
Forward Primer (10 μM)	1 μL
Reverse Primer (10 μM)	1 μL
DNA Template (plasmid or colony PCR)	<200 ng
Nuclease-Free Water	Adjust to 50 μL

Note: DNA templates were derived from plasmid extraction products or direct colony lysates (see "Colony PCR" section). To avoid contamination, all reagents were prepared on ice, using filtered pipette tips, and the reaction tubes were placed on a pre-cooled PCR machine before initiating the program. Primer Sequences (Selected Representative Primers, Designed According to RFC#10 Standard) All primers were validated using Primer-BLAST to avoid dimer or hairpin formation and eliminate EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI restriction sites (except those retained for cloning purposes): PCR Program Using a PCR machine, the program was as follows:

Step	Temperature	Time	Cycles
Initial Denaturation	98 °C	5 min	1
Denaturation	98 °C	10 s	30 cycles
Annealing	55 °C	5 s
Extension	72 °C	1 min/kb
Final Extension	72 °C	8 min	1
Hold	4 °C	∞	—

Note: Extension time is calculated based on fragment length (e.g., 2.0 kb fragment → 2 min). Annealing temperature was uniformly set at 55 °C for all target fragments. Amplified products were verified by 1% agarose gel electrophoresis (containing GelRed). Colony PCR (Rapid Screening of Positive Clones)

To rapidly screen transformants, direct colony PCR was used without plasmid extraction:

1. Pick a single colony with a sterile toothpick and gently touch it into 20 μL of sterile water in a 1.5 mL centrifuge tube. Vortex to mix;
2. Heat the bacterial solution in a 95 °C metal bath for 5 min to lyse cells and release DNA;
3. Cool on ice for 30 s, then use 1 μL of lysate as PCR template;
4. After amplification, run gel electrophoresis and select positive bands (matching target fragment size) for subsequent sequencing validation.

PCR Product Purification

All target PCR products were purified using the Magen Gel Extraction Kit (China) as follows:

1. Under UV light, cut out the target band, minimizing non-target gel material, and weigh (100 mg ≈ 100 μL volume);
2. Add 3 volumes of Buffer GDP (e.g., 150 mg gel + 450 μL GDP);
3. Incubate at 55 °C for 10–15 min, gently inverting every 3–5 min until the gel is fully dissolved;
4. Cool to room temperature, transfer to HiPure DNA Mini Column, centrifuge at 12,000 × g for 30–60 s, discard flow-through;
5. Add 600 μL Buffer DW2 (containing ethanol), centrifuge 30–60 s, discard flow-through;
6. Add another 300 μL Buffer DW2, centrifuge at 12,000 × g for 2 min to remove ethanol completely;
7. Place column into a new 1.5 mL centrifuge tube, add 30 μL Elution Buffer (or sterile water) to membrane center, let stand for 1 min;
8. Centrifuge at 12,000 × g for 1 min to collect DNA solution;
9. Measure concentration and purity using NanoDrop 2000 (A260/A280 > 1.8), store at –20 °C.

Gel Electrophoresis

PCR amplification of key gene fragments was verified by agarose gel electrophoresis, with band sizes matching expectations:

• VvRHM: 2025 bp
• UGT73B2: 1449 bp
• Cm1,2RhaT: 1356 bp
• ThF3′H: 1374 bp
• CPR: 1029 bp
• F4′OMT: 1917 bp
• pCadC-mRFP: 1078 bp
• pCspA-mRFP: 738 bp

All products showed no non-specific bands, confirming accurate cloning and amplification.

Plasmid Construction and Cloning

We used the pET28a(+) vector (Novagen/Merck Millipore, USA) to construct two main expression modules:

1. Hesperetin Synthesis Module: ThF3′H, CPR, and MpOMT S142V were cloned into pET28a(+) via NdeI/XhoI restriction sites to form a polycistronic expression unit under T7 promoter control. Recombinant plasmids were transformed into E. coli BL21(DE3) via heat shock (42 °C, 1 min) and selected on LB agar plates containing 100 μg/mL kanamycin.
2. Neo Hesperidin Synthesis Module: UGT73B2, VvRHM-NRS, and Cm1,2RhaT were assembled as a polycistronic sequence based on B0034 RBS, also cloned into pET28a(+) via NdeI/XhoI sites and transformed into E. coli BL21(DE3), with selection on LB plates containing kanamycin.

For biosensor and safety system construction, pSB1A3 vector was used:

• pCadC-mRFP: pCadC promoter fused upstream of mRFP using In-Fusion HD Cloning Kit (Takara Bio);
• pBAD-MazF: MazF lethal gene downstream of pBAD promoter;
• pCspA-MazF: MazF downstream of pCspA cold-inducible promoter.

The above constructs were transformed into E. coli BL21 or DH5α, with selection on LB plates containing 100 μg/mL ampicillin.

Specific plasmid construction and transformation steps:

To construct high-precision, seamless, multi-fragment assembled engineering plasmids (such as pCadC-mRFP, pBAD-MazF, pCspA-MazF, and the VvRHM-UGT73B2-PcadBA-Cm1,2RhaT composite module), this study employed seamless cloning. This method relies on efficient homologous recombination based on 15 bp homology arms at DNA ends, requiring no restriction enzymes or ligase, enabling single or triple fragment assembly simultaneously, significantly improving efficiency and accuracy.

1. Preparation of Target Fragments and Vector PCR Amplification of Insert Fragments and Linearized Vector

All insert fragments (e.g., pCadC, mRFP, MazF, pBAD, pCspA) and linearized pET28a+ vector were amplified by high-fidelity PCR. Primers were designed to include 15 bp homology arms at both ends, precisely matching the vector termini.

Vector Linearization

Using pET28a+ as a universal vector, it was linearized by PCR: using terminal primers, the linearized vector backbone was directly amplified (removing the antibiotic resistance gene intermediate sequence), ensuring 15 bp homology arms at the ends. Products were gel-extracted with concentration ≥20 ng/μL.

Recombination Reaction Mixture (10 μL)

Prepare the following reaction mixture in sterile PCR tubes (room temperature operation):

Component	Volume
Pre-mix	2 μL
Linearized Vector	50–100 ng
Insert Fragment 1	50–150 ng
Insert Fragment 2 (if applicable)	50–150 ng
Insert Fragment 3 (if applicable)	50–150 ng
Nuclease-Free Water	Adjust to 10 μL

Recombination Program

Gently mix the components, briefly centrifuge, and place in PCR machine for the following program:

Step	Temperature	Time
Annealing/Extension	50 °C	15 min

Transformation and Screening

Transformation of Competent Cells

1. Add 10 μL of reaction product to 50 μL of E. coli DH5α or E. coli BL21(DE3);
2. Incubate on ice for 30 min;
3. Heat shock at 42 °C for 90 s;
4. Immediately place on ice for 2 min;
5. Add 500 μL of liquid medium (without antibiotics), recover with shaking at 37 °C, 200 rpm for 1 h.

Plate Screening

1. Plate 100 μL of recovered culture on LB agar plates with appropriate antibiotics, incubate at 37 °C for 12–16 h;
2. Pick 10–15 single colonies and inoculate into LB liquid medium with antibiotics, culture overnight at 37 °C.

Positive Clone Verification

1. Colony PCR: Use vector universal primers to amplify insert fragments, confirm correct size;
2. Plasmid Extraction: Extract plasmids using Tiangen Plasmid Mini Kit;

Sequencing Verification: Send samples to Eurofins Genomics for bidirectional sequencing to confirm:

2. Complete correctness of insert fragment sequence;
3. No mutations or deletions;
4. Seamless connection at homology arm regions, no extra bases.

All strains were preserved long-term at –80 °C with 25% (v/v) glycerol as cryoprotectant.

Hesperetin Production

Inoculate constructed strains overnight culture at 1% (v/v) into 25 mL LB medium containing 100 μg/mL kanamycin. Culture at 37 °C, 180 rpm. When OD₆₀₀≈0.6, add 20 g/L naringenin (dissolved in DMSO), reduce temperature to 25 °C, and continue fermentation for 24 h.

Mix 0.5 mL fermentation broth with 0.5 mL methanol, vortex, centrifuge at 13,500 rpm for 5 min. Filter supernatant through 0.22 μm membrane for HPLC analysis.

Neo Hesperidin Production

Same cultivation method. Three hours after induction, add 3 g/L hesperetin (dissolved in DMSO), continue fermentation at 25 °C, 180 rpm for 48 h.

Sample processing and HPLC detection methods were the same as above.

HPLC Analysis of Flavonoids

Instrumentation and Conditions Agilent 1200 Series HPLC equipped with diode array detector (DAD) and ZORBAX Eclipse Plus C18 column (250 × 4.6 mm, 5 μm). Hesperetin Detection Conditions:

• Detection Wavelength: 280 nm
• Flow Rate: 1.0 mL/min
• Column Temperature: 30 °C
• Injection Volume: 10 μL

Mobile Phase:

• Phase A: 100% Acetonitrile (HPLC grade)
• Phase B: 0.5% (v/v) Acetic Acid Solution

Gradient Elution Program:

Time (min)	% B
0–12	85 → 75
12–17	75 (isocratic)
17–20	75 → 50
20–30	50 → 25
30–35	25 → 5
35–40	5 → 85 (column re-equilibration)

Neo Hesperidin Detection Conditions:

• Detection Wavelength: 290 nm
• Column Temperature: 35 °C

Mobile Phase:

• Phase A: 0.1% Trifluoroacetic Acid Water Solution
• Phase B: 0.1% Trifluoroacetic Acid Methanol Solution

Gradient Elution Program:

Time (min)	% B
0–0.1	10
0.1–10.0	10 → 40
10.0–20.0	40 → 60
20.0–22.0	60 → 10
22.0–25.0	10 (isocratic)

Standard Retention Times:

• Naringenin: 17.5 min
• Eriodictyol: 14.1 min
• Hesperetin: 18.1 min
• Neo Hesperidin: 16.2 min
• Hesperetin-7-O-glucoside: 18.2 min

Compounds were identified by comparison of retention time and UV spectrum, quantified using external standard method.

Characterization of Biosensors and Induction Systems

Acid-Responsive Promoter (pCadC) Testing

The pCadC-mRFP reporter strain was cultured in LB medium at pH 5.8 and 7.3. Fluorescence intensity (Ex: 584 nm / Em: 607 nm) and OD₆₀₀ were measured simultaneously using FlexStation 3 microplate reader at intervals, normalized fluorescence (fluorescence/OD₆₀₀) was calculated.

Low pH Induction of Neo Hesperidin Synthesis

Engineered strain VvRHM-UGT73B2-PcadBA-Cm1,2RhaT was cultured to OD₆₀₀ = 1.2, then transferred to medium at pH 5.8 or 7.3 with 3 g/L hesperetin added. Samples were taken every 12 h for analysis.

Arabinose Induction System

The pBAD-mRFP strain was inoculated into LB medium containing 0–0.5% L-arabinose, cultured at 37 °C, fluorescence and OD₆₀₀ were monitored.

Arabinose-Inducible Lethal System

The pBAD-MazF engineered strain was inoculated into M9 medium containing 0.2% arabinose and 0.4% glucose (glucose suppresses background expression), OD₆₀₀ was monitored.

Cold-Shock Induction System

The pCspA-mRFP strain was cultured at 16 °C, 25 °C, 30 °C, and 37 °C for 12 h, normalized fluorescence was measured.

Cold-Shock Inducible Lethal System

The pCspA-MazF engineered strain was cultured at 16 °C in LB medium containing ampicillin, growth curve was monitored.

Neo Hesperidin Production Condition Optimization

To maximize yield, BL21-Neohes was induced for fermentation (24 h) at 25 °C, 30 °C, and 37 °C with 3 g/L hesperetin added.

Statistical Analysis

All experimental data were analyzed using GraphPad Prism. Results are expressed as mean ± standard deviation (SD), with at least three biological replicates per group.

• Comparison between two groups: Student's t-test
• Comparison among multiple groups: One-way ANOVA + Tukey's post-hoc test

p < 0.05 was considered statistically significant.