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Metabolic and genetic factors affecting the productivity of pyrimidine nucleoside in Bacillus subtilis.

Zhu H, Yang SM, Yuan ZM, Ban R - Microb. Cell Fact. (2015)

Bottom Line: Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively.Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L.Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China. zhuhui0505@hotmail.com.

ABSTRACT

Background: Cytidine and uridine are produced commercially by Bacillus subtilis. The production strains of cytidine and uridine were both derivatives from mutagenesis. However, the exact metabolic and genetic factors affecting the productivity remain unknown. Genetic engineering may be a promising approach to identify and confirm these factors.

Results: With the deletion of the cdd and hom genes, and the deregulation of the pyr operon in Bacillus subtilis168, the engineered strain produced 200.9 mg/L cytidine, 14.9 mg/L uridine and 960.1 mg/L uracil. Then, the overexpressed prs gene led to a dramatic increase of uridine by 25.9 times along with a modest increase of cytidine. Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively. Moreover, the overexpression of the pyrH gene increasesd the yield of cytidine by 40%, along with a modest augments of uridine and uracil. Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L.

Conclusions: The deregulation of the pyr operon and the overexpression of the prs, pyrG and pyrH genes all contribute to the accumulation of pyrimidine nucleoside compounds in the medium. Among these factors, the overexpression of the pyrG and pyrH genes can particularly facilitate the production of cytidine. Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.

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The biosynthetic pathway of pyrimidine nucleotide in B. subtilis.
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Fig1: The biosynthetic pathway of pyrimidine nucleotide in B. subtilis.

Mentions: Bacillus subtilis is able to synthesize the uridine 5'-monophosphate (UMP) de novo. The excess UMP can be further converted to terminal metabolites (cytidine, uridine and uracil), which could then be secreted out of the cell (Figure 1). The pyrimidine nucleotide biosynthetic (pyr) operon of B. subtilis contains 10 genes. The first gene of the pyr operon encodes a bifunctional protein PyrR which is the regulator protein for pyr operon and a uracil phosphoribosyl transferase [1]. The second gene, pyrP, encodes a uracil permease. The remaining eight genes encode the six enzymes involved in the de novo biosynthesis of UMP [2]. The expression of the pyr operon is regulated by transcriptional attenuation mechanism involving PyrR. The PyrR protein is mainly activated by UMP and the regulating elements are three specific binding loops (BL1, BL2 and BL3) on the nascent pyr mRNA. The combination of the PyrR protein and BLs disrupts the antiterminator, permitting the formation of terminator hairpin and leading to the reduced expression of the downstream genes [3,4]. The resulting high intracellular concentration of UMP would strongly inhibit the transcription of the pyr operon.Figure 1


Metabolic and genetic factors affecting the productivity of pyrimidine nucleoside in Bacillus subtilis.

Zhu H, Yang SM, Yuan ZM, Ban R - Microb. Cell Fact. (2015)

The biosynthetic pathway of pyrimidine nucleotide in B. subtilis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403831&req=5

Fig1: The biosynthetic pathway of pyrimidine nucleotide in B. subtilis.
Mentions: Bacillus subtilis is able to synthesize the uridine 5'-monophosphate (UMP) de novo. The excess UMP can be further converted to terminal metabolites (cytidine, uridine and uracil), which could then be secreted out of the cell (Figure 1). The pyrimidine nucleotide biosynthetic (pyr) operon of B. subtilis contains 10 genes. The first gene of the pyr operon encodes a bifunctional protein PyrR which is the regulator protein for pyr operon and a uracil phosphoribosyl transferase [1]. The second gene, pyrP, encodes a uracil permease. The remaining eight genes encode the six enzymes involved in the de novo biosynthesis of UMP [2]. The expression of the pyr operon is regulated by transcriptional attenuation mechanism involving PyrR. The PyrR protein is mainly activated by UMP and the regulating elements are three specific binding loops (BL1, BL2 and BL3) on the nascent pyr mRNA. The combination of the PyrR protein and BLs disrupts the antiterminator, permitting the formation of terminator hairpin and leading to the reduced expression of the downstream genes [3,4]. The resulting high intracellular concentration of UMP would strongly inhibit the transcription of the pyr operon.Figure 1

Bottom Line: Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively.Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L.Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China. zhuhui0505@hotmail.com.

ABSTRACT

Background: Cytidine and uridine are produced commercially by Bacillus subtilis. The production strains of cytidine and uridine were both derivatives from mutagenesis. However, the exact metabolic and genetic factors affecting the productivity remain unknown. Genetic engineering may be a promising approach to identify and confirm these factors.

Results: With the deletion of the cdd and hom genes, and the deregulation of the pyr operon in Bacillus subtilis168, the engineered strain produced 200.9 mg/L cytidine, 14.9 mg/L uridine and 960.1 mg/L uracil. Then, the overexpressed prs gene led to a dramatic increase of uridine by 25.9 times along with a modest increase of cytidine. Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively. Moreover, the overexpression of the pyrH gene increasesd the yield of cytidine by 40%, along with a modest augments of uridine and uracil. Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L.

Conclusions: The deregulation of the pyr operon and the overexpression of the prs, pyrG and pyrH genes all contribute to the accumulation of pyrimidine nucleoside compounds in the medium. Among these factors, the overexpression of the pyrG and pyrH genes can particularly facilitate the production of cytidine. Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.

Show MeSH
Related in: MedlinePlus