Enhancement of cytidine production by coexpression of gnd , zwf , and prs genes in recombinant Escherichia coli CYT15
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ORIGINAL RESEARCH PAPER
Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15 Haitian Fang • Xixian Xie • Qingyang Xu Chenglin Zhang • Ning Chen
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Received: 2 September 2012 / Accepted: 4 October 2012 / Published online: 16 October 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-aD-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine pro
duction by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli. Keywords Cytidine production Co-expression Escherichia coli Metabolic flux Phosphoribosylpyrophosphate synthetase 6-Phosphoglucose dehydrogenase 6-Phosphogluconate dehydrogenase
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s10529-012-1068-3) contains supplementary material, which is available to authorized users. H. Fang X. Xie Q. Xu C. Zhang N. Chen (&) Metabolic Engineering Laboratory, College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin 300457, China e-mail: [email protected] H. Fang (&) College of Agriculture, Ningxia University, Yinchuan, China e-mail: [email protected]
Cytidine is an important drug precursor and food ingredient. Since the 1970s, high-level cytidine production by Bacillus subtilis, B. pumilus, B. licheniformis, Escherichia coli and other organisms has been reported (Turnbough and Switzer 2008; Koo et al. 2011). Some of these processes have been patented. The general strategy used to generate a cytidineoverproducing bacterial strain involves modulating the regulation of key pathways. Several microorganisms (e.g., E. coli and B. subtilis) have been modified for cytidine production by standard mutagenesis methods and selected for the ability to grow on toxic cytidine analogues (Lee et al. 2010). Commercial cytidine production was developed in E. coli by a combination of standard mutagenesis,
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rational metabolic engineering, and genetic engineering of cytidine biosynthetic genes to improve fermentation. To increase cytidine production further, additional targets for metabolic engineering have been identified. For example, pr
