The gene sequence encoding alkane monooxygenase (AlkM) was synthesized and codon-optimized for E. coli. This sequence was cloned into the pET23b plasmid using NdeI and XhoI restriction sites to create a recombinant plasmid (Azenta, USA). The recombinant plasmid was designed to express the AlkM gene under the control of the constitutive T7 promoter. After sequence verification (Genewiz, China), the plasmid was extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmid was then transformed into E. coli DH5α for plasmid preservation and E. coli BL21 for protein expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C.

Tools used: Gnina [https://github.com/gnina/gnina](https://github.com/gnina/gnina)

Protein visualization tool: Schrödinger Maestro

Command: `gnina -r *.pdb -l *.sdf --autobox_ligand *.sdf -o *.sdf.gz`

Affinity: Binding affinity refers to the strength of the interaction between a single biomolecule (e.g., protein or DNA) and its ligand/binding partner (e.g., drug or inhibitor). Binding affinity is typically measured and reported using the equilibrium dissociation constant (KD), which evaluates the strength of bimolecular interactions and ranks their intensity. The smaller the KD value, the greater the binding affinity of the ligand for its target. Conversely, a larger KD value indicates weaker attraction and binding between the target molecule and the ligand.

The coding gene sequence of BsLac was synthesized and codon-optimized for E. coli. The sequence was cloned into the pET23b plasmid using NdeI and XhoI restriction sites, resulting in a recombinant plasmid (Azenta, USA). The downstream coding gene is constitutively expressed under the T7 promoter. After sequence verification (Qingke, China), the recombinant plasmid was extracted using a plasmid extraction kit (Tiangen, China). The plasmid was then transformed into E. coli DH5α for storage and BL21 for expression. The engineered strain was cultured in LB medium containing 50 μg/mL ampicillin (Amp) at 37°C.

The BsLac coding gene and a truncated ice nucleation protein (INP) sequence were synthesized and positioned upstream of BsLac. The fusion protein INP-BsLac coding sequence was cloned into the pET23b plasmid via NdeI and XhoI restriction sites, resulting in a recombinant plasmid (Genewiz, USA). Similarly, recombinant engineered strains based on BL21 were obtained.

The wild-type BL21, control strain (containing only the pET23b empty plasmid), and BsLac-overexpressing strains were inoculated at a 1:100 ratio into fresh LB medium containing 50 μg/mL ampicillin. After overnight incubation at 37°C, 2 mL of bacterial culture was collected. The cultures were centrifuged at 8000 rpm for 10 minutes, and the bacterial pellet was resuspended in PBS (pH 7.4). Ultrasonic disruption was performed using a Biosafer1000 ultrasonic homogenizer (Saifei) under ice bath conditions (75 W, 1s on, 3s off, 20 min) to obtain crude enzyme extracts. Laccase activity was measured using the ABTS method. ABTS was incubated with BsLac enzyme solution in a water bath at 30°C, and absorbance changes at 420 nm were measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific, USA). The reaction system consisted of 930 μL Na2HPO4-citrate buffer (50 mM, pH 3.2), 20 μL ABTS (10 mM), and 50 μL enzyme solution. One enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of ABTS per minute per mL of solution.

To determine the optimal temperature, laccase activity was measured with ABTS as a substrate in 50 mM Na2HPO4-citrate buffer (pH 3.2) at different temperatures (20-60°C). The effect of pH on laccase activity was assessed at 30°C across a pH range of 2.0 to 6.0. Glycine-HCl buffer was used for pH 2.0, and Na2HPO4-citrate buffer was used for pH 3.0–6.0.

Laccase activity was measured using ABTS as the substrate at a wavelength of 480 nm. The reaction mixture was prepared as follows: 1 mL of 5 mM ABTS, 0.5 mL of cell suspension, and 1.5 mL of 0.1 M acetate buffer (pH 5.0). The 3 mL mixture was reacted at 30°C in the dark. Enzyme activity was measured at 480 nm by spectrophotometry, and enzyme activity units were calculated per gram of bacterial dry weight.

The synthetic iaaM and iaaH genes (polycistronic structure with RBS B0034 between genes) were inserted into the pET23b vector (Azenta, USA) at the NdeI and XhoI cloning sites. After sequence verification, the plasmid was transformed into E. coli BL21.

The IPA pathway requires three enzymes to convert L-tryptophan into IAA in three steps. Tryptophan transaminase (aro8) converts tryptophan into indole-3-pyruvate, which is then converted into indole-3-acetaldehyde by indole-3-pyruvate decarboxylase (kdc). Indole-3-acetaldehyde is finally converted to indole-3-acetic acid by aldehyde dehydrogenase (puuc). The aro8 and kdc genes are derived from yeast, while puuc is from E. coli. The synthetic aro8, kdc, and puuc genes (polycistronic structure, RBS B0034 between genes) were inserted into the pET23b vector (Azenta, USA) at NdeI and XhoI cloning sites. After sequence verification, the plasmid was transformed into E. coli BL21.

The engineered and control strains were activated overnight in LB medium at 37°C with shaking at 180 rpm. The following day, 1 mL of culture was transferred to 50 mL of fresh medium and incubated at 37°C, 180 rpm. The flasks were wrapped in aluminum foil to prevent IAA degradation by light. After centrifuging 1.5 mL of bacterial culture (10000 rpm, 1 min), 1 mL of the supernatant was collected. For IAA detection, 1 mL of the supernatant was mixed with 1 mL of Salkowski reagent (15 mL 0.5 M FeCl₃, 300 mL concentrated sulfuric acid, 500 mL distilled water). The mixture was incubated in the dark for 30 minutes, turning pink, indicating the presence of IAA. The absorbance was measured at 530 nm using spectrophotometry, with absorbance proportional to IAA concentration. The IAA content in the bacterial cultures was quantified using a standard curve prepared with known concentrations of IAA.

To evaluate the influence of the supernatant from engineered strains on seed germination, four types of seeds were selected: water spinach, soybeans, wheat, and black wheat. For each type, 200 seeds were divided into an experimental group and a control group, with 100 seeds in each. The experimental group was treated with the supernatant from IAM pathway-engineered strains, while the control group was treated with water.

The engineered strain was cultured overnight in LB medium at 37°C and 180 rpm. The following day, 1 mL of the bacterial culture was transferred to 50 mL of fresh medium, wrapped in aluminum foil to avoid light degradation. After 12 hours of incubation, the culture was centrifuged to collect the supernatant. Seeds in the experimental group were soaked in this supernatant for 12 hours, while control seeds were soaked in water. After soaking, the seeds were transferred to germination trays with water. Seed germination was observed after two days.

To assess the impact of engineered strain supernatant on root growth under petroleum stress, healthy seeds of water spinach and soybeans were selected. Six seeds of each type were randomly divided into two groups: the experimental group (3 seeds) and the control group (3 seeds). The experimental group was treated with the supernatant from an IAM pathway-engineered strain.

The engineered strain was cultured overnight in LB medium, and the next day, 1 mL of bacterial culture was transferred to 50 mL of fresh LB medium wrapped in aluminum foil. After 12 hours of incubation at 37°C and 180 rpm, the culture was centrifuged, and the supernatant was collected. Seeds in the experimental group were soaked in the supernatant for 12 hours, while the control group seeds were soaked in water. After soaking, the seeds were transferred to soil containing trace amounts of petroleum and root growth was observed.

The sequences encoding AlkS and PAlkB were obtained through gene synthesis and cloned into the pSB1A3 vector (using XbaI and SpeI restriction sites), positioned upstream of the mRFP gene coding sequence. The expression of the transcription factor is regulated by the constitutive J23100 promoter. The recombinant plasmid was transformed into E. coli DH5α.

The alkane biosensor strain was inoculated into LB medium at a ratio of 1:100 and cultured overnight at 37°C. The following day, it was transferred to fresh M9 medium (containing 50 μg/mL ampicillin: Na2HPO4, 3.0 g/L; KH2PO4, 0.5 g/L; NaCl, 1.0 g/L; NH4Cl, 1.0 g/L; MgSO4, 5.0 mM; CaCl2, 0.1 mM; supplemented with 10 g/L glucose) at a 1:50 ratio. Meanwhile, n-dodecane was dissolved in anhydrous ethanol containing 1% Tween 80 (200 mg/L) and mixed thoroughly using a magnetic stirrer. Subsequently, solutions of n-dodecane at different concentrations were added to the culture medium. Induction was conducted at 37°C for 20 hours. A 1 mL aliquot of the culture was taken, and OD600 values and fluorescence were measured using a microplate reader (excitation wavelength 584 nm, emission wavelength 607 nm). The normalized fluorescence ratio (Fluorescence/OD600) was calculated.

Data were analyzed and graphed using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). For comparisons of multiple groups, one-way ANOVA followed by Tukey’s post-hoc test was used for difference analysis. For comparisons of two groups, a two-tailed Student’s t-test was performed. A p-value of less than 0.05 was considered statistically significant.

(1) Gang, S.; Sharma, S.; Saraf, M.; Buck, M.; Schumacher, J. Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiellaby LC-MS/MS and the Salkowski Method. Bio Protoc 2019, 9 (9), e3230. DOI: 10.21769/BioProtoc.3230 From NLM.