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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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Pyrimidine and pyrimidine nucleoside accumulation by B. subtilis strains after 72 h fermentation. Results are the average of three replicates with error bars indicating standard error from the mean (TD02, TD12, TD13, TD231, TD232 and TD33).
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Fig2: Pyrimidine and pyrimidine nucleoside accumulation by B. subtilis strains after 72 h fermentation. Results are the average of three replicates with error bars indicating standard error from the mean (TD02, TD12, TD13, TD231, TD232 and TD33).

Mentions: In order to observe the effects of the related genetic modification on cytidine and uridine synthesis separately, we blocked the reaction from cytidine to uridine by deleting 151 bp coding sequences of the cdd gene in B. subtilis 168 N and obtained the cdd gene deficient strain B. subtilis TD01 (Additional file 1: Figure S1). Subsequently, in order to improve the supplement of aspartate for the pyrimidine nucleotide biosynthesis, we deleted 827 bp coding sequences of the hom gene in the strain TD01 and obtained the strain B. subtilis TD02 (Additional file 1: Figure S2). The shake-flask culture experiments demonstrated that the strain TD02 could accumulate cytidine and uracil in detectable level in medium while no uridine was detected (Figure 2).Figure 2


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)

Pyrimidine and pyrimidine nucleoside accumulation by B. subtilis strains after 72 h fermentation. Results are the average of three replicates with error bars indicating standard error from the mean (TD02, TD12, TD13, TD231, TD232 and TD33).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Pyrimidine and pyrimidine nucleoside accumulation by B. subtilis strains after 72 h fermentation. Results are the average of three replicates with error bars indicating standard error from the mean (TD02, TD12, TD13, TD231, TD232 and TD33).
Mentions: In order to observe the effects of the related genetic modification on cytidine and uridine synthesis separately, we blocked the reaction from cytidine to uridine by deleting 151 bp coding sequences of the cdd gene in B. subtilis 168 N and obtained the cdd gene deficient strain B. subtilis TD01 (Additional file 1: Figure S1). Subsequently, in order to improve the supplement of aspartate for the pyrimidine nucleotide biosynthesis, we deleted 827 bp coding sequences of the hom gene in the strain TD01 and obtained the strain B. subtilis TD02 (Additional file 1: Figure S2). The shake-flask culture experiments demonstrated that the strain TD02 could accumulate cytidine and uracil in detectable level in medium while no uridine was detected (Figure 2).Figure 2

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