Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Chemicals
2.2. Strains and Plasmids
2.3. Protein Expression and Purification
2.4. Enzymatic Activity Assays
2.5. Production of Arabinosides from Sucrose or G6P
2.6. Effect of NADP+ Concentrates on Ru5P Accumulation
2.7. Effect of Phosphate Concentrations on the Accumulation of Intermediate Metabolite
2.8. Analytical Methods
3. Results and Discussion
3.1. Design of the Multi-Enzymatic Cascade for Arabinoside Production from Sucrose and Nucleobase
3.2. Enzyme Selection of NP
3.3. Feasibility Verification of Ara-A Production from Sucrose and Adenine
3.4. Optimization of Ara-A Production from Sucrose and Adenine
3.5. Production of Ara-A from Sucrose and Adenine under Optimal Conditions
3.6. Production of Other Arabinosides through In Vitro Multi-Enzymatic Cascade Equipped with Different NPs
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
Ara-A | Vidarabine |
Ara-G | Arabinofuranosylguanine |
Ara-C | Cytarabine |
Ara-U | Spongouridine |
Ara-I | Hypoxanthine arabinofuranoside |
NADP+ | Nicotinamide adenine dinucleotide phosphate |
IPTG | Isopropyl β-D-1-thiogalactopyranoside |
MTT | Methylthiazolyldiphenyl-tetrazolium bromide |
PES | Phenazine ethosulfate |
SP | Sucrose phosphorylase |
PGM | Phosphoglucomutase |
G6PD | Glucose 6-phosphate 1-dehydrogenase |
6PGDH | 6-phosphogluconate dehydrogenase |
API | D-arabinose 5-phosphate isomerase |
PPM | Phosphopentomutase |
NP | Nucleoside phosphorylase |
PNP | Purine nucleoside phosphorylase |
UP | Uridine phosphorylase |
TP | Thymidine phosphorylase |
PyNP | Pyrimidine nucleoside phosphorylase |
RK | Ribokinase |
TPNOX | H2O-forming NADPH oxidase |
G1P | Glucose 1-phosphate |
G6P | Glucose 6-phosphate |
GlcA6P | 6-phosphogluconate |
Ru5P | Ribulose 5-phosphate |
A5P | Arabinose 5-phosphate |
A1P | Arabinose 1-phosphate |
HPLC | High-performance liquid chromatography |
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Name | Enzyme | Source | Substrate Spectra |
---|---|---|---|
KlPNP | Purine nucleoside phosphorylase | Klebsiella | Ara-A, Ara-G, and Ara-I |
AaPNP | Purine nucleoside phosphorylase | Alicyclobacillus acidoterrestris | Ara-A, Ara-G, and Ara-I |
BsPNP | Purine nucleoside phosphorylase | Bacillus subtilis | Ara-A, Ara-G, and Ara-I |
BlPNP | Purine nucleoside phosphorylase | Bacillus licheniformis | Ara-A, Ara-G, and Ara-I |
EcPNP | Purine nucleoside phosphorylase | E. coli | Ara-U |
EcUP | Uridine phosphorylase | E. coli | Ara-U |
EcXP | Purine nucleoside phosphorylase 2 | E. coli | Ara-G |
EcTP | Thymidine phosphorylase | E. coli | Ara-U |
Name | Enzyme | Source | Conditions Required for Activity | Reference |
---|---|---|---|---|
EcPGM | Phosphoglucomutase | E. coli | pH 8.0, Mg2+ | [26] |
EcG6PD | Glucose-6-phosphate 1-dehydrogenase | E. coli | / | |
Ec6PGDH | 6-phosphogluconate dehydrogenase | E. coli | pH 8.0, NADP+, Mg2+ | [27] |
EcAPI | D-arabinose 5-phosphate isomerase | E. coli | pH 8.5; Zn2+ inhibited | [21] |
BcPPM | Phosphopentomutase | Bacillus cereus | pH 8.0, glucose 1,6-bisphosphate dependent, Mn2+ | [28] |
TPNOX | NADPH oxidase | Lactobacillus brevis | pH 7.5, NADPH | [20] |
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Liu, Y.; Yang, E.; Zhang, X.; Liu, X.; Tang, X.; Wang, Z.; Wang, H. Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade. Biomolecules 2024, 14, 1107. https://doi.org/10.3390/biom14091107
Liu Y, Yang E, Zhang X, Liu X, Tang X, Wang Z, Wang H. Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade. Biomolecules. 2024; 14(9):1107. https://doi.org/10.3390/biom14091107
Chicago/Turabian StyleLiu, Yuxue, Erchu Yang, Xiaojing Zhang, Xiaobei Liu, Xiaoting Tang, Zhenyu Wang, and Hailei Wang. 2024. "Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade" Biomolecules 14, no. 9: 1107. https://doi.org/10.3390/biom14091107