Whole‐cell biosynthesis of cytarabine by an unnecessary protein‐reduced Escherichia coli that coexpresses purine and uracil phosphorylase

• Published in: Biotechnology and bioengineering Vol. 119; no. 7; pp. 1768 - 1780
• Main Authors: Ping, Li, Ruxian, Jing, Mengping, Zhou, Pei, Jia, Zhuoya, Li, Guosheng, Liu, Zhenyu, Wang, Hailei, Wang
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2022
• Subjects: Adenine; Biosynthesis; Catalysis; Catalysts; Chemical synthesis; Conversion; Cytarabine; E coli; Enzymes; Escherichia coli; gene deletion; Genetic modification; Genomes; Interference; Phosphorylase; Phosphorylases; Protein purification; Proteins; Substrates; unnecessary proteins; Uracil; whole‐cell catalysis; cytarabine; unnecessary proteins; whole-cell catalysis; Escherichia coli; gene deletion
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.28098

Currently, whole‐cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole‐cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large‐scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole‐cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole‐cell catalysis. An efficient strategy to lessen interference from unnecessary proteins for biocatalysis was developed in this study. Li Ping and coworkers have conducted a large‐scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of unnecessary proteins on cytarabine production, which is equivalent to partial purification of the enzymes including purified purine phosphorylase and pyrimidine nucleoside phosphorylase within cells. By using the strategy, the conversion rate of whole‐cell catalysis was very close to that of pure‐enzyme catalysis.