Building of a synthetic phosphoketolase pathway that effectively catabolizes a number of carbon sources to acetyl-CoA

Quantum-chemical evaluation for PK from Actinobacteria Bifidobacterium (BbPK)

Molecular docking of substrate to BbPK

Phosphoketolase choice

Protein engineering of BbPK

To acquire full mutations, oligonucleotide primers have been designed with the degenerate codon NNK, which cowl virtually all mutations with solely 96 clones. Therefore, a complete of three,264 clones have been screened in opposition to 34 single-site saturation mutation libraries. Every single-site saturation mutant library was generated primarily based on PCR. The PCR product was degraded the template with DpnI restriction endonuclease, after which remodeled into E. coli BL21 (DE3) competent cells for library development. Every colony was incubated in 200 μL of LB medium for twenty-four h to plateau at 37°C after which transferred to 1 mL of the identical medium for protein expression. The cells, which induced by IPTG (isopropyl-β-D-thiogalactopyranoside) and cultured in a single day at 16°C, have been harvested by centrifugation. The bacterial pellet was washed and resuspended in response buffer (50 mM potassium phosphate buffer, 5 mM GALD or 20 mM DHA (pH 7.4)). After 3 h of response at 37°C, the supernatant was collected by centrifugation for detection of substrate or product.

For GALD, we decided the exercise of the mutants by detecting the discount of substrate. The detection methodology was as follows: added 120 μL chromogenic reagent to 60 μL pattern and heated at 90°C for 15 min. Subsequently, the residual substrate focus was measured spectrophotometrically at 650 nm. The chromogenic reagent: 1.5 g diphenylamine was dissolved in 100 mL acetic acid, after which 1.5 mL pure sulfuric acid was added.

For DHA, we screened for extremely lively mutants by detecting the product formaldehyde. The formaldehyde detection methodology was as follows: 40 μL pattern was combined with 160 μL chromogenic reagent, after which heated at 60°C for 10 min. Subsequently, formaldehyde manufacturing was measured spectrophotometrically at 440 nm, and 100 mL chromogenic reagent (pH 6.0) comprises 25 g ammonium acetate, 3 mL acetic acid, and 0.25 mL acetylacetone resolution.

Exercise assay and kinetic properties of PK and mutants

The usual response combination (100 μL) contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 1 mM ThDP, 10 mM GALD (DHA or D-EUS), 1 mM ADP, 0.2 mg mL⁻¹ AckA, 5 U hexokinase, 2.5 U Glucose-6-phosphate dehydrogenase, 1 mM NADP⁺, and 10 mM glucose. Roughly 0.5 mg mL⁻¹ PK was added into the response system. The reactions carried out at 37°C. NADPH was detected spectrophotometrically at 340 nm. Enzyme kinetics with GALD (DHA or D-EUS) as substrate have been decided in assays with GALD (DHA or D-EUS) concentrations of 0–110 mM. Kinetic parameters kcat and Km have been decided by measuring the preliminary velocities of the enzymic response and curve-fitting in accordance with the Michaelis–Menten equation, utilizing GraphPad Prism 5 software program.

Bacterial strains and progress situation

Escherichia coli DH5α pressure (TransGenTM) was grown at 37°C in LB medium for gene cloning and different DNA manipulations. E. coli BL21 (DE3) (TransGenTM) was grown at 37°C or 16°C in 2YT medium for protein expression. Antibiotics for choice functions have been used with 100 μg mL⁻¹ spectinomycin, 100 μg mL⁻¹ ampicillin, 34 μg mL⁻¹ chloramphenicol, or 100 μg mL⁻¹ kanamycin.

Enzyme exercise of BbPK for DHA, L-EUS, and D-EUS

The coding gene of phosphoketolase from Actinobacteria Bifidobacterium was ligated into the expression vector pET-28a through NdeІ and XhoI restriction websites. E. coli BL21(DE3) cells carrying recombinant plasmid have been inoculated into 5 mL LB (Luria Broth) medium with Kanamycine (100 μg mL⁻¹) and cultured in a single day at 37°C, after which scaled as much as 800 mL 2YT medium (16 g L⁻¹ Tryptone, 10 g L⁻¹ yeast extract, 5 g L⁻¹ NaCl) containing Kanamycine (100 μg mL⁻¹). Gene expression was induced by including IPTG to a closing focus of 0.5 mM when OD₆₀₀ reached 0.6. The cell cultures continued to develop in a single day at 16°C earlier than being harvested by centrifugation at 6,000×g after which was resuspended in 50 mL lysis buffer (50 mM potassium phosphate buffer (pH 7.4), 5 mM MgSO₄, 0.5 mM ThDP). The bacterial pellet was lysed through the use of a high-pressure homogenizer (JNBIO, China), and the cell particles was eliminated by centrifugation at 10,000×g for 60 min at 4°C. The soluble protein pattern was loaded onto a nickel affinity column (GE Healthcare), which was rinsed with 50 mL wash buffer (50 mM potassium phosphate buffer (pH 7.4), 5 mM MgSO₄, 0.5 mM ThDP, and 50 mM imidazole) after which eluted with 20 mL elution buffer (50 mM potassium phosphate buffer (pH 7.4), 5 mM MgSO₄, 0.5 mM ThDP, and 200 mM imidazole). The eluted protein was concentrated and dialyzed in opposition to lysis buffer (50 mM potassium phosphate buffer (pH 7.4), 5 mM MgSO₄, and 0.5 mM ThDP) by ultrafiltration with an Amicon Extremely centrifugal filter machine (Millipore, USA) with a 30 kDa molecular-weight cutoff. The protein focus was decided utilizing a BCA Protein Assay Reagent Equipment (Pierce, USA) with BSA as the usual.

Exercise of PKs on DHA was decided with 1 mg mL⁻¹ purified recombinant protein in 200 μL response mixtures. The response system comprised 10 mM DHA, 1 mM ThDP, 5 mM MgSO₄, and 50 mM phosphate buffer (pH 7.4). After incubation at 37°C for 0.5 h, the response was stopped by including 200 μL of acetonitrile. Acetyl-phosphate in samples is decided by chromogenic and liquid chromatography, the by-product formaldehyde was confirmed by GC-MS.

Chromogenic detection of acetyl-phosphate (AcP): add 100 μL hydroxylamine hydrochloride resolution (2 M, pH 6.5) to 100 μL pattern, conduct at 30°C for 10 min. Then add 200 μL of chromogenic resolution, which is ready with 10 mL FeCl₃.6H₂O (FeCl₃.6H₂O powder dissolved in 0.1 M hydrochloric acid, 5% (m/v)), 10 mL trichloroacetic acid (15% (m/v)), and 10 mL HCl (4 M), conduct at 30°C for five min, then detect the absorbance at 505 nm.

HPLC detection for AcP: add equal quantity of 5% sulfuric acid to the pattern for fully decomposing AcP into acetic acid. Acetic acid was then detected by HPLC. HPLC circumstances: column, Aminex HPX-87H (Bio-Rad); detection wavelength, 210 nm; cellular part, 5 mM sulfuric acid; stream fee, 0.6 mL min⁻¹; pattern quantity, 20 μL; column temperature, 40°C.

Formaldehyde detection by GC-MS. Pattern derivatization: 100 μL 2.4-dinitrophenylhydrazine resolution was added to 100 μL samples, the combination was carried out at 60°C for 60 min at midnight. Then added 400 μL of n-hexane to the combined resolution, centrifuge at 5,000×g for two min. Separated the higher resolution after which added applicable quantity of anhydrous sodium sulfate powder, centrifuged at 10,000×g for 10 min. Separated the higher resolution and was detected by GC-MS. GC-MS circumstances: Electron ionization (EI) GC-MS analyses have been carried out with a mannequin 7890A GC (Agilent) with a DB-5 fused silica capillary column (30 cm size, 0.25 mm interior diameter, 0.25 μm movie thickness) coupled to an Agilent 7200 Q-TOF mass selective detector. Injections have been carried out by a mannequin 7683B autosampler. The GC oven was programmed from 160°C (held for 1 min) to 240°C at 10°C min⁻¹, after which held for five min; the injection port temperature was 250°C, and the switch line temperature was 280°C. The provider gasoline, ultra-high purity helium, flowed at a relentless fee of 1.2 mL min⁻¹. For full-scan information acquisition, the MS scanned from 35 to 550 atomic mass items. Information evaluation for GC-MS was carried out with Mass Hunter software program (Agilent, USA) and NIST Database.

The exercise of PKs on L-EUS and D-EUS have been decided with 1 mg mL⁻¹ purified recombinant protein in 200 μL response mixtures. The response system comprised 10 mM L-EUS or D-EUS, 1 mM ThDP, 5 mM MgSO₄, and 50 mM phosphate buffer (pH 7.4). After incubation at 37°C for 0.5 h, the response was stopped by including 200 μL of acetonitrile. AcP in samples was detected by chromogenic and liquid chromatography. The by-product glycoaldehyde was confirmed by GC-MS. Chromogenic and liquid chromatography detection of AcP have been similar as DHA.

Glycoaldehyde was confirmed by GC-MS. Samples have been freeze-dried, then 60 μL of 0.2 M PFBOA resolution was added and combined, the response was carried out at 30°C for 90 min. A complete of 300 μL of n-hexane was added and centrifuge at 5,000×g for two min. Separated the higher resolution after which added applicable quantity of anhydrous sodium sulfate powder, centrifuged at 10,000×g for 10 min. Separated 100 μL higher resolution, then 30 μL N-Methyl-N-(trimethylsilyl)-trifluoroacetamide with 1% trimethylchlorosilane was added and combined, the response system carried out at 30°C for 30 min. The product was detected by GC-MS. GC-MS circumstances: EI GC-MS analyses have been carried out with a mannequin 7890A GC (Agilent) with a DB-5 fused silica capillary column (30 cm size, 0.25 mm interior diameter, 0.25 μm movie thickness) coupled to an Agilent 7200 Q-TOF mass selective detector. Injections have been carried out by a mannequin 7683B autosampler. The GC oven was programmed from 60°C (held for 1 min) to 100°C at 5°C min⁻¹, to 300°C at 25°C min⁻¹ after which held for five min; the injection port temperature was 250°C, and the switch line temperature was 280°C. The provider gasoline, ultra-high purity helium, flowed at a relentless fee of 1.2 mL min⁻¹. For full-scan information acquisition, the MS scanned from 35 to 550 atomic mass items. Information evaluation for GC-MS was carried out with Mass Hunter software program (Agilent, USA) and NIST Database.

Exercise of PKs on E4P, D-ETS, D-GCD, and DHAP have been decided with 1 mg mL⁻¹ of purified recombinant protein in 200 μL response mixtures. The response system included substrate (10 mM E4P, D-ETS, D-GCD, or DHAP), 1 mM ThDP, 5 mM MgSO₄, and 50 mM phosphate buffer (pH 7.4). After incubation at 37°C for 0.5 h, the response was stopped by including 200 μL of acetonitrile. AcP in samples was detected by chromogenic and liquid chromatography. Chromogenic and liquid chromatography detection of AcP have been similar as DHA.

Exercise of PKs on erythrulose-4-phosphate (Eu4P) have been decided with 1 mg mL⁻¹ purified recombinant protein in 200 μL response mixtures. The response system included 10 mM Eu4P, 0.5 mg mL⁻¹ RpiB, 1 mM ThDP, 5 mM MgSO₄, and 50 mM phosphate buffer (pH 7.4). After incubation at 37°C for 0.5 h, the response was stopped by including 200 μL of acetonitrile. AcP in samples was detected by chromogenic and liquid chromatography. Chromogenic and liquid chromatography detection of AcP have been similar as DHA.

Expression, purification, and enzyme kinetics of PKs from totally different species

The PKs coding genes from totally different species have been ligated into the expression vector pET-28a through NdeІ and XhoI restriction websites. All genes have been expressed in BL21 (DE3) and purified on the Ni-NTA column. Giant-scale purification (800 mL) usually produced about 50 mg enzyme. The protein focus was decided utilizing the BCA Protein Assay Reagent Equipment (Pierce, USA) with BSA as the usual.

Dedication of kinetics of PKs on GALD, DHA, L-EUS, and D-EUS. The usual response combination (200 μL) contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 1 mM ThDP, 10 mM GALD (or, DHA, L-EUS, D-EUS), 1 mM ADP, 0.2 mg mL⁻¹ AckA, 1 U hexokinase, 0.5 U glucose-6-phosphate dehydrogenase, 1 mM NADP⁺, and 10 mM glucose. Varied PKs (0.25 mg mL⁻¹) from totally different species have been added into the response system. The reactions carried out at 37°C. The manufacturing of NADPH was detected at 340 nm. Enzyme kinetics with DHA, L-EUS, and D-EUS as substrates have been decided in assays with concentrations of 0–110 mM. Kinetic parameters have been decided from triplicate experiments utilizing GraphPad Prism 5 (GraphPad Software program, USA).

Dedication of kinetics of PKs on Xu5P. The usual response combination (200 μL) contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 1 mM ThDP, 5 mM Xu5P, 0.05 mg mL⁻¹ TIM, 1 U glycerophosphate dehydrogenase, and 1 mM NAD⁺. Varied PKs (0.1 mg mL⁻¹) from totally different species have been added into the response system. The reactions carried out at 37°C. The manufacturing of NADH was detected at 340 nm.

Dedication of kinetics of PKs on F6P. The usual response combination (200 μL) contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 1 mM ThDP, 5 mM F6P, 0.3 mg mL⁻¹ erythrose-4-phosphate dehydrogenase, and 1 mM NAD⁺. Varied PKs (0.1 mg mL⁻¹) from totally different species have been added into the response system. The reactions carried out at 37°C. The manufacturing of NADPH was detected at 340 nm.

Expression, purification, and enzyme kinetics of phosphatases from totally different species

The phosphatases coding genes of various species have been ligated into the expression vector pET-28a through NdeІ and XhoI restriction websites. All genes have been expressed in BL21 (DE3) and purified on the Ni-NTA column. Giant-scale purification (800 mL) usually produced about 5 to 50 mg enzyme. The protein focus was decided utilizing a BCA Protein Assay Reagent Equipment (Pierce, USA) with BSA as the usual.

The kinetics of phosphatases on F6P, Xu5P, E4P, G3P, and DHAP have been decided by monitoring the manufacturing of fructose, xylulose, ETS, GCD, and DHA by HPLC. The usual response combination (100 μL) contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM F6P (or Xu5P, E4P, G3P, DHAP). Varied phosphatases (0.1 mg mL⁻¹) from totally different species have been added into the response system. The reactions carried out at 37°C for 0.5 to 2 h, and 100 μL acetonitrile was added to the samples to terminate the reactions, after which analyzed by HPLC.

HPLC situation for GCD or DHA: column, Aminex HPX-87H (Bio-Rad); detection wavelength, 210 nm; cellular part, 5 mM sulfuric acid; stream fee, 0.6 mL min⁻¹; pattern quantity, 20 μL; column temperature, 40°C.

HPLC detection for fructose, xylulose, and erythrose. Pattern derivatization: Response samples (50 μL) have been combined with an answer of O-benzylhydroxylamine hydrochloride (50 μL, 0.14 mmol mL⁻¹) (pyridine: methanol: water = 33:15:2). After incubation at 50°C for 60 min, samples have been diluted in methanol (100 μL) and straight analyzed by HPLC chromatography. HPLC situation: column, X-BridgeTM C18, 5 μm, 4.6 × 250 mm column from Waters (Milford, USA); pattern quantity, 30 μL; solvent system, (A) aqueous trifluoroacetic acid (TFA) (0.1% (v/v) and (B) TFA (0.095% (v/v)) in CH₃CN/H₂O (4:1), gradient elution from 20% to 60% B in 16 min; stream fee, 1 mL min⁻¹; detection wavelength, 215 nm; column temperature, 35°C.

Formaldehyde circulation system verification

Comparability of PK-GALS formaldehyde recycling system and PK-FLS formaldehyde recycling system: The 100 μL response system contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 2 mg mL⁻¹ PK, 2 mg mL⁻¹ GALS or FLS, 10 mM DHA, and 1 mM ThDP. The reactions carried out at 37°C for two h, and 100 μL sulfuric acid (5%) was added to the samples to terminate the reactions. Acetic acid was then detected by HPLC. HPLC circumstances: column, Aminex HPX-87H (Bio-Rad); detection wavelength, 210 nm; cellular part, 5 mM sulfuric acid; stream fee, 0.6 mL min⁻¹; pattern quantity, 20 μL; column temperature, 40°C.

Acetic acid and DHA detection methodology is similar as above. Formaldehyde was detected by chromogenic methodology. Chromogenic detection of formaldehyde is as follows: dilute the pattern to the suitable focus (0.1 to 1 mM), add 80 μL chromogenic resolution to 120 μL of the diluted pattern. The response carried out at 60°C for 10 min and centrifugal at 12,000×g for five min. Take 150 μL to measure the absorbance at 414 nm. Calculate the focus of formaldehyde within the pattern in accordance with the usual curve. Chromogenic resolution is ready as follows: Dissolve 250 g of ammonium acetate in 900 mL of ddH₂O, then add 30 mL of acetic acid and a pair of.5 mL of acetylacetone, alter the pH of the answer to six with acetic acid, and eventually add ddH₂O to a quantity of 1 L.

Course of evaluation of the APK pathway for F6P

The 1 mL response system contained 50 mM potassium phosphate buffer (pH 7.5) 5 mM MgSO₄, 2 mg mL⁻¹ PK, 0.5 mg mL⁻¹ EcHAD, 1 mg mL⁻¹ Ps-LRHI, 1 mM ThDP, 10 mM F6P. The reactions carried out at 37°C for 10 h. Samples have been taken out each 2 h for evaluation. The reactions have been terminated by heating at 95°C for five min, after which cooled all the way down to 37°C. Added the 5 U alkaline phosphatase, and carried out at 37°C for 4 h, equal quantity acetonitrile was used to terminate the response. Fructose, D-ETS, D-EUS, GALD, and acetic acid have been detected by HPLC.

Pattern derivatization: Response samples (50 μL) have been combined with an answer of O-benzylhydroxylamine hydrochloride (50 μL, 0.14 mmol mL⁻¹) (pyridine: methanol: water = 33:15:2). After incubation at 50°C for 60 min, samples have been diluted in methanol (100 μL) and straight analyzed by HPLC. HPLC circumstances: column, X-Bridge TM C18, 5 μm, 4.6 × 250 mm column from Waters (Milford, USA); pattern quantity, 30 μL; cellular part, solvent system (A) TFA (0.1% (v/v) and (B) TFA (0.095% (v/v)) in CH₃CN/H₂O (4:1), gradient elution from 20% to 60% B in 16 min; stream fee, 1 mL min⁻¹; detection wavelength, 215 nm; column temperature, 35°C.

Course of evaluation of the APK pathway for Xu5P

The 1 mL response system contained 50 mM potassium phosphate buffer (pH 7.5) 5 mM MgSO₄, 2 mg mL⁻¹ PK, 1 mg mL⁻¹ CpHAD, 0.1 mg mL⁻¹ TIM, 2 mg mL⁻¹ FLS, 1 mM ThDP, and 10 mM Xu5P. The reactions carried out at 37°C for 10 h, and samples have been taken out each 2 h for evaluation. The reactions have been terminated by heating at 95°C for five min, then cooled all the way down to 37°C. Added the 5 U alkaline phosphatase, and carried out at 37°C for 4 h, equal quantity acetonitrile was used to terminate the response. D-xylulose, GCD, DHA, and acetic acid have been detected by HPLC. Formaldehyde was detected by chromogenic methodology as earlier than.

HPLC detection for D-xylulose, GCD and DHA. Pattern derivatization: Response samples (50 μL) have been combined with an answer of O-benzylhydroxylamine hydrochloride (50 μL, 0.14 mmol mL⁻¹) (pyridine: methanol: water = 33:15:2). After incubation at 50°C for 60 min, samples have been diluted in methanol (100 μL) and straight analyzed by HPLC chromatography. HPLC circumstances: column, X-BridgeTM C18, 5 μm, 4.6 × 250 mm column from Waters (Milford, USA); pattern quantity, 30 μL; cellular part, solvent system (A) TFA (0.1% (v/v) and (B) TFA (0.095% (v/v)) in CH₃CN/H₂O (4:1), gradient elution from 20% to 60% B in 16 min; stream fee, 1 mL min⁻¹; detection wavelength, 215 nm; column temperature, 35°C. HPLC circumstances of acetic acid have been the identical as earlier than.

The APK pathway for the utilization of C1-C4 carbon sources

The APK pathway for D-EUS and GALD. The 200 μL response system contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 2 mg mL⁻¹ PK, 1 mM ThDP, 10 mM GALD, or 5 mM D-EUS. The reactions carried out at 37°C in a single day.

The APK pathway for FALD and DHA. The 200 μL response system contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO₄, 2 mg mL⁻¹ PK, 2 mg mL⁻¹ FLS, 1 mM ThDP, 20 mM FALD, or 10 mM DHA. The reactions carried out at 37°C in a single day.