期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 119, 期 7, 页码 1768-1780出版社
WILEY
DOI: 10.1002/bit.28098
关键词
cytarabine; Escherichia coli; gene deletion; unnecessary proteins; whole-cell catalysis
资金
- Excellent Scientific and Technological Innovation Team of Henan Normal University, China [5101049170501]
- National Science Foundations of China [U160411067]
In this study, the efficiency of whole-cell catalysis for cytarabine synthesis was improved through gene editing to reduce interference from unnecessary proteins (UNPs). The obtained mutant showed increased enzyme activities and conversion rates compared to the parental strain.
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.
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