Presentation Information
[P01-065]Engineering Escherichia coli for the production of pyrimidines
○Yun-Peng Chao1, Chung-Jen Chiang2 (1. Department of Chemical Engineering, Feng Chia University (Taiwan), 2. Department of Medical Laboratory Science and Biotechnology, China Medical University (Taiwan))
Keywords:
pyrimidines,metabolic engineering,genetic engineering
[Purpose] A synthetic nucleoside derivative has many applications in the pharmaceutical industry, including the synthesis of antiviral and antitumor drug precursors. Pyrimidine nucleoside analog drugs are of particular importance for their potential to combat life-threatening viruses. In Escherichia coli, the de novo biosynthesis of pyrimidine comprises several reaction steps. It starts with bicarbonate (HCO3-) and the amino group from glutamine (Gln), and also requires aspartate (Asp) and ribose-5-phosphate (R5P). In this study, the overproduction of pyrimidines in E. coli was achieved by manipulating central metabolism and the pyrimidine synthesis pathway.
[Method] The pyr operon of Bacillus subtilis 168 was reassembled by PCR. It resulted in three synthetic operons: one containing pyrAA and pyrAB, another containing pyrB and pyrC, and the other containing pyrK, pyrD, pyrF, and pyrE. In addition, the endogenous genes involving prsA, rpiA, zwf, and pgl in the pentose phosphate (PP) pathway were assembled into two artificial operons. The E. coli producer strains were then obtained by integrating each synthetic operon.
[Results] The E. coli strain with the operon containing pyrAA and pyrAB was able to produce a detectable level of uracil and uridine. The production level increased in E. coli carrying the entire pyr operon of B. subtilis after introducing two additional synthetic operons. The two synthetic operons consisting of endogenous genes were further introduced into the strain. This enabled the strain to produce more uracil than uridine. Bicarbonate serves as the precursor. A significant increase in uracil production was obtained for the strain with the engineered PP pathway when bicarbonate was supplemented. The attenuated expression of pgi in the strain showed a positive effect on uracil, comparable to that of bicarbonate. The pyrimidine synthesis pathway requires amino acids, Gln and Asp. These two amino acids are derived from glutamate (Glu). As a result, Glu supplementation improved uracil production in the strain with a titer reaching 1.2 g/L. Uracil phosphoribosyltransferase (encoded by upp) plays a physiological role in the pyrimidine salvage. Uridine-5-monophosphate (UMP) phosphatase (encoded by umpH) is a ribonucleotide monophosphatase with a preference for UMP. Without the supplement of bicarbonate and Glu, the overexpression of upp and umpH improved the uracil production in the cell.
[Consideration] The pyrimidine synthesis pathway in E. coli is stringently regulated, and the product overflow occurs when genes involved in the pathway are upregulated. The engineered PP pathway leads to the formation of 5’-phosphoribosyl pyrophosphate (PRPP), an intermediate metabolite required for the pyrimidine synthesis. This engineered pathway works efficiently after a bicarbonate supplement. It suggests that the PRPP level is limited in the pyrimidine synthesis pathway. Moreover, the requirements for Gln and Asp are met by the Glu supplement.
[Conclusion] In this study, the limiting steps in the pyrimidine synthesis pathway were identified in a systematic way. We also found several genes that function to interconvert UMP, uridine, and uracil. It provides useful information for the rational design of E. coli for the production of various pyrimidine-derived metabolites. This work is currently underway in our lab.
[Method] The pyr operon of Bacillus subtilis 168 was reassembled by PCR. It resulted in three synthetic operons: one containing pyrAA and pyrAB, another containing pyrB and pyrC, and the other containing pyrK, pyrD, pyrF, and pyrE. In addition, the endogenous genes involving prsA, rpiA, zwf, and pgl in the pentose phosphate (PP) pathway were assembled into two artificial operons. The E. coli producer strains were then obtained by integrating each synthetic operon.
[Results] The E. coli strain with the operon containing pyrAA and pyrAB was able to produce a detectable level of uracil and uridine. The production level increased in E. coli carrying the entire pyr operon of B. subtilis after introducing two additional synthetic operons. The two synthetic operons consisting of endogenous genes were further introduced into the strain. This enabled the strain to produce more uracil than uridine. Bicarbonate serves as the precursor. A significant increase in uracil production was obtained for the strain with the engineered PP pathway when bicarbonate was supplemented. The attenuated expression of pgi in the strain showed a positive effect on uracil, comparable to that of bicarbonate. The pyrimidine synthesis pathway requires amino acids, Gln and Asp. These two amino acids are derived from glutamate (Glu). As a result, Glu supplementation improved uracil production in the strain with a titer reaching 1.2 g/L. Uracil phosphoribosyltransferase (encoded by upp) plays a physiological role in the pyrimidine salvage. Uridine-5-monophosphate (UMP) phosphatase (encoded by umpH) is a ribonucleotide monophosphatase with a preference for UMP. Without the supplement of bicarbonate and Glu, the overexpression of upp and umpH improved the uracil production in the cell.
[Consideration] The pyrimidine synthesis pathway in E. coli is stringently regulated, and the product overflow occurs when genes involved in the pathway are upregulated. The engineered PP pathway leads to the formation of 5’-phosphoribosyl pyrophosphate (PRPP), an intermediate metabolite required for the pyrimidine synthesis. This engineered pathway works efficiently after a bicarbonate supplement. It suggests that the PRPP level is limited in the pyrimidine synthesis pathway. Moreover, the requirements for Gln and Asp are met by the Glu supplement.
[Conclusion] In this study, the limiting steps in the pyrimidine synthesis pathway were identified in a systematic way. We also found several genes that function to interconvert UMP, uridine, and uracil. It provides useful information for the rational design of E. coli for the production of various pyrimidine-derived metabolites. This work is currently underway in our lab.
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