Component	Volume
Restriction Endonuclease	1 μL
Buffer	2 μL
DNA	1 μg
ddH2O	up to 20 μL

1. Prepare the system of Restriction Endonuclease cleaving DNA.
2. Restriction Endonucleases are incubated at 37°C for 1 h except for BamH I, which is incubated at 30°C. The mixture is further digested by reaction at 80°C for 5 min and stored at 4°C.

Reagent	Volume
Phanta Taq Master Mix(2x)	25 μL
Primer F(10 μM)	2 μL
Primer R(10 μM)	2 μL
Template DNA(Plasmid)	＜20 ng
ddH2O	Up to 50 μLg

Temperature (℃)	Time	Cycles
95	3 min
95	15 sec	25-35
55-72	15 sec
72	30~60 sec/kb
72	5 min

1. Column equilibrium: Add 500 μL of equilibrium solution BL to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. Cut the single band of target DNA from the agarose gel, place it into a clean centrifuge tube, and weigh it.
3. Add moderate volume of solution PC to the glue block and place it in a water bath at 50°C for 10 min, during which the centrifuge tube is gently turned up and down to ensure that the glue block is fully dissolved.
4. Add the solution obtained in the previous step to an adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.
5. Add 600 μL of rinsing solution PW to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.
6. Repeat step 5.
7. The adsorption column CB2 is put into the collection tube and centrifuged at 12000 rpm for 2 min. The adsorption column is left at room temperature for several minutes and allowed to dry thoroughly.
8. Put the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane by hanging drops, place it at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to collect the DNA solution.

1. Configuration of homologous recombination reaction system.
2. Use a pipettor to gently suck and mix (do not shake and mix violently), and briefly centrifugal the reaction solution to the bottom of the tube.
3. React at 37°C for 30 min, then drop to 4°C or immediately cool on ice.
One Step Cloning reaction system:

Component	Volume
Linearized plasmid	2 μL
The fragment to be inserted	12 μL
5 × CE II Buffer	4 μL
Exnase II	2 μL

1.Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.
2. Measure 0.5 g agarose, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 × TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.
3. Add Ultra GelRed (10,000 ×) to the solution.
4.Pour the solution into the gel casting tray.
5. After the gel has solidified, pull out the comb.
6. Place the gel in the electrophoresis chamber with enough 1x TAE buffer.
7. Transfer the mixture to the well on the gel with a pipette.
8. Power on, run at 130 V for 20 minutes.

1. Column equilibrium: Add 500 μL of equilibrium solution BL to the adsorption column CP3, centrifuge at 12000 rpm for 1min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. Take 1 to 5 mL of the bacterial solution cultivated overnight, add it to the centrifuge tube, centrifuge at 12000 rpm for 1 min, and remove the supernatant as much as possible.
3. Add 250 μL of solution P1 (with added RNaseA) to the centrifuge tube containing the bacterial precipitate to completely suspend the bacterial precipitate.
4. Add 250 μL solution P2 to the centrifuge tube and gently turn it up and down for 6 to 8 times to fully lyse the thallus.
5. Add 350 μL of solution P3 to the centrifuge tube and immediately turn it gently up and down 6 to 8 times, at which point a white flocculent precipitate appears. The samples are centrifuged at 12000 rpm for 10 min.
6. Transfer the supernatant collected in the previous step to the adsorption column CP3 using a pipetting gun. After centrifugation at 12000 rpm for 30~ 60 sec, the waste liquid in the collection tube is poured out and the adsorption column CP3 is placed into the collection tube.
7. Add 600 μL of rinsing solution PW to the adsorption column CP3 (absolute ethanol has been added), centrifuge at 12000rpm for 30~60 sec, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.
8. Repeat steps.
9. The adsorption column CP3 is put into the collection tube and centrifuged at 12000 rpm for 2 min.
10. Place the adsorption column CP3 in a clean centrifuge tube, add 50 to 100 μL of elution buffer EB to the middle part of the adsorption membrane, place it at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.

Preparation of chemically transformation competent cells

1. Add 1 mL of overnight culture seed medium to a shaker flask containing 100 mL of LB medium and culture in a shaker at 37°C to about OD₆₀₀=0.5.
2. Centrifugal harvesting, 6000 rpm, 5 min.
3.Add 10 mL of pre-cooled 100 mM CaCl₂ for resuspension.
4. Apply cold compress on ice for 20 min.
5. Centrifugal harvesting, 4000 rpm, 10 min.
6. Add 4 mL of pre-cooled 100 mM CaCl₂ and resuspend again.
7. Apply cold compress on ice for 20 min.
8. Centrifugation, 4000 rpm, 10 min.
9. Add 1mL of pre-cooled 100 mM CaCl₂ and 15% glycerol mixed solution.
10. Aliquot in pre-cooled 1.5 mL centrifuge tubes and store at -80°C.

Preparation of electrocompetent cells

1. Single colony of plasmid-introduced strain is inoculated into 5mL LB and cultured for 12~14 h as seed medium.
2. Take 1~2 mL of seed medium into a shaker containing 100mL of LB medium and culture until the OD₆₀₀ value reaches 0.4~0.5.
3. Transfer the solution to a 50mL centrifuge tube and apply cold compress on ice for 20 min.
4.Centrifuge at 4°C, 4000 rpm for 10 min.
5. Resuspend the precipitation by 40 mL of pre-cooled sterile water and centrifuge at 4°C, 4,000 rpm for 10 min.
6. Resuspend the precipitation by another 40 mL of pre-cooled sterile water and centrifuge at 4°C, 4000 rpm for 10 min.
7. Take 20 mL of pre-cooled glycerol with a volume fraction of 10% for resuspension and centrifuge at 4°C, 4000 rpm for 10 min.
8. Finally, resuspend with 300 μL of pre-cooled glycerol with a volume fraction of 10%, aliquot in 1.5 mL centrifuge tubes, and store at -80°C.

Chemically transformation

1.Take competent cells from -80°C refrigerator and put it on ice.
2. When the competent cells thaw, add 10 μL DNA ligation product or 2 μL plasmid per tube, place the mixture on ice for 30 min.
3. Heat shock at 42°C for exactly 45 seconds.
4.Put the 1.5 mL tubes back on ice for 3 min.
5. Add 800 μL LB fluid medium without antibiotics into the 1.5 mL tubes and then culture in the shaker incubator at 37°C for an hour.
6. Extract 100-200 μL bacteria liquid, transferred to LB solid medium with antibiotics and spread plates.
7.Place plates upside down and incubate at 37°C overnight.

Electroformation

1. 100 μL of competent cells and 5μL of each plasmid are mixed and added to a precooled Electric rotor cup and placed on ice for 5 min.
2. Turn on the electrometer and set the program:

Condition	Numerical value
Voltage	2500 V
Capacitance	25 μF
Resistance	200 Ω
Cuvette	2 mm

3. Clean the water on the outer wall of the electric rotating cup and conduct electric rotating.
4. After electroporation, 800 μL LB medium is added Immediately, transfer to a 1.5 mL centrifuge tube, and incubate in a shaker at 37°C for 1 h.
5. Centrifuge at 4000 rpm for 3 min, 650 μL of supernatant is aspirated, and the remainder is resuspended on LB solid medium with corresponding antibiotics and incubated for 20 h.

Component	The volume (mL) of each component required to prepare different volumes of SDS-PAGE separation gels
8% glue	5	10	15	20	30	50
ddH2O	2.4	4.7	7.1	9.5	14.2	23.7
30%Acr-bis (29:1)	1.3	2.7	4.0	5.3	8.0	13.3
Lower glue buffer (4X)	1.25	2.5	3.75	5.0	7.5	12.5
10% gel polymerization catalyst	0.05	0.1	0.15	0.2	0.3	0.5
TEMED	0.003	0.006	0.006	0.012	0.018	0.03

1. After the bacteria are collected and crushed, the ultrasonic crushing machine has a power of 30%, and the working time is 2 sec with an interval of 3 sec for a total of 10 min.
2. After crushing, the supernatant is centrifuged at 6000 rpm for 10 min to obtain the crude enzyme solution.
3. Add the corresponding proportion of gravity nickel column to the obtained crude enzyme solution, place it on ice, shake it thoroughly at 200 rpm for 20~30 min, so that the target enzyme can combine with the nickel column.
4. Gravity elution: Prepare centrifuge tubes filled with 700 μl Coomassie Brilliant Blue staining solution.The gradient concentration of imidazole (15 ~30 mM) is added to the purification column.The miscellaneous proteins are eluted. The protein elution will be completed until the Coomassie brilliant blue staining solution do not change color after adding the protein eluent to the Coomassie brilliant blue staining solution.
5. Purification: The gravity column is rinsed with a high concentration of imidazole (100~300 mM), and the eluate is collected as the target enzyme.
6. The collected and purified enzyme is transferred to an ultrafiltration tube for desalting and centrifuged at 4500 rpm for 30 min.
7. Add PBS to the ultrafiltration tube, wash, centrifuge at 4500 rpm for 30 min, and repeat twice.
8. Concentrate the pure enzyme to less than 500 μL and collect the enzyme solution.

The colony was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.1 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD₆₀₀=0.6~0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na₂HPO₄ and 100 mM NaH₂PO₄.
React the prepared system at 30℃ and 200 rpm.

System for whole-cell catalytic production of 5-aminovaleramide

Component	Content
Cell concentration	OD600=5
L-Lysine (100 g/L)	1 mL
PBS	to 10 mL

System for whole-cell catalytic production of 5-aminovalerate

Component	Content
Cell concentration	OD600=5
L-Lysine (100 g/L)	1 mL
PBS	to 10 mL

System for whole-cell catalytic production of 5-hydroxyvalerate

Component	Content
Cell concentration	OD600=40
5-aminovalreate (100 g/L)	250 μL
glucose (500 g/L)	20 μL
CaCO3	0.1 g
α-ketoglutarate (100g/L)	250 μL
PBS	to 5 mL

System for whole-cell catalytic production of 1,5-pentanediol

Component	Content
Cell concentration	OD600=20
5-Hydroxyvaleric (100 g/L)	250 μL
glucose (560 g/L)	90 μL
MnSO4·H2O (50 g/L)	75 μL
PBS	to 5 mL

The 5 mL seed medium NE of NT1003 contained 10 g/L of (NH₄)₂SO₄ and 5 g/L of DL-monosodium glutamate, 10 g/L Yeast Extract, 0.655 g/L K₂HPO₄·3H₂O, 0.55 g/L C₃H₃NaO₃, 5 g/L Cane sugar, 0.3 g/L Methionine, 0.3 g/L Threonine, 1 g/L MgSO₄·7H₂O, 0.032 g/L FeSO₄·7H₂O and 0.077 g/L MnSO₄·H₂O. We used the final concentration of 100 μg/mL Amp, 50 μg/mL Kan, 25 μg/mL Cm in our experiments. Shaker cultures were performed in 50 mL shaker tubes containing 5 mL NE medium.

The fermentation medium NR of NT1003 contained 10 g/L of (NH₄)₂SO₄ and 5 g/L of Tryptone, 2 g/L Yeast Extract, 0.5 g/L KCl, 1.6 g/L MgSO₄·7H₂O, 23.125 g/L MoPO₅Na,10 g/L CaCO₃, 0.086 g/L ZnSO₄·7H₂O, 0.077 g/L CuSO₄, 0.032 g/L MnSO₄·H₂O, 20 g/L glucose, 0.036 g/L Methionine, 0.036 g/L Threonine, 0.036 g/L VB₁, 0.006 g/L NAM and 0.036 mg/L biotin. Shake flask fermentation was carried out in 500mL conical flasks containing 30mL NR medium. Main cultures were inoculated at an initial OD₆₀₀=0.2 from overnight precultures in NE medium. 1ml of the bacterial solution was added to the shaker. The bacteria were grown at 37°C and 200 rpm. Expression of pathway enzymes was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the cells density reached OD₆₀₀=1.0. At the same time, the shaker temperature was changed to 30°C, and the bacteria were still grown at 200 rpm.

The culture medium of the fermenter was basically the same as the NR medium, except that the initial concentration of glucose was changed to 50 g/L, and other conditions were unchanged. Primary seed cultures were still performed using 50 mL shaker tubes containing 10 mL NE medium. Secondary seed cultures were still grown using 2000 mL conical flasks containing 250 mL. When the cells density reached OD₆₀₀=2.0, all of them were transferred to the fermenter. The initial cells density in fermenter was OD₆₀₀=0.2. The initial ventilation volume was 0.6 vvm. The temperature was controlled to 30°C by the instrument. The pH was controlled at 7.0 and regulated with ammonia. The supplementary culture medium contained 400 g/L ammonium sulfate and 600 g/L glucose, and the fermenter was controled the dissolved oxygen level at 20% to 30%. Samples were taken every 3 hours to detect the cells density, glucose content, lysine content and ammonia nitrogen content.

The cells density was detected by the absorbance value at 600 nm by UV spectrophotometer.

SBA was used to detect the content of glucose and lysine. After calibration with 100 g/L glucose and lysine standard solution, the glucose and lysine content of the sample was detected.

Quantification of acetic acid, 5-hydroxyvalerate, 1,5-pentanediol and other organic acids were carried out by using High performance liquid chromatography (HPLC) equipped with an Aminex HPX-87H Column (300*7.8 mm). HPLC used 8 mM sulfuric acid as the mobile with a flow rate of 0.6 mL/min, and detection via RID.

Amino acid derivatization of the samples was first performed using the GPC method. The samples were quantitatively analyzed by high-performance liquid chromatography (HPLC). The Column was EF-C18 5 μL 120Å 4.6*250nm, the column temperature was 40℃, the mobile phase was 80% acetonitrile and 108 g/L sodium acetate dissolved in acetonitrile, and gradient washing was used. The flow rate was 1.0 mL/min, and VWD absorption detection was performed at 254 nm.