An enzymatic assay for high-throughput screening of cytidine-producing microbial strains.

Huina Dong,Ning Li,Yongfei Liu,Feiran Li,Xin Zu,Dawei Zhang,Gotthard Kunze

doi:10.1371/journal.pone.0121612

Abstract

Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more).

Highlights

Cytidine is an industrially important precursor for the production of some medicines, such as cytarabine hydrochloride and its intermediate ancitabine hydrochloride [1], 2'fluoro-2'-deoxycytidine and 2'fluoro-2'-deoxy-arabinocytidine [2]
The coding region of cdd gene from B. subtilis was expressed in E. coli using pET28a expression system
The cytidine deaminase (CDA) protein was purified as the His-tagged fusion protein (18.48 kDa), which resulted in a CDA preparation with an approximately 95% purity (Fig. 1) judged by SDS-PAGE

Summary

Introduction

Cytidine is an industrially important precursor for the production of some medicines, such as cytarabine hydrochloride and its intermediate ancitabine hydrochloride [1], 2'fluoro-2'-deoxycytidine and 2'fluoro-2'-deoxy-arabinocytidine [2]. An Enzymatic Assay for Cytidine to all the PLOS ONE policies on sharing materials and data. The enzymatic steps in de novo biosynthetic pathways of pyrimidine nucleotides such as UTP, CTP and dCTP are regulated in-vivo by feedback inhibition of key enzymes and by repression or attenuation of enzyme synthesis, by accumulation of end products or other metabolites [3, 4]. To accumulate a large amount of cytidine, cells must be resistant to the feedback regulation, which means cells have to be metabolically modified. Several microorganisms (e.g. Escherichia coli, Bacillus subtilis) [5,6,7] have been modified for cytidine production by standard mutagenesis methods using ultraviolet radiation, diethyl sulfate treatment or lowenergy ion mutations. The positive mutant strains are selected by toxic cytidine analogs

Methods

Results

Conclusion