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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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Relative transcriptional levels of mRNA in the recombinant strains. The mRNA expression levels of WT strain is regarded as 1(blank). The relative mRNA expression levels of recombinant strains (filled) are compared with WT strain. (A) Relative transcriptional levels of the pyrR gene. ΔpyrR: the pyrR gene was deleted. (B) Relative transcriptional levels of the prs gene. prs+: the prs gene was overexpressed. (C) Relative transcriptional levels of the pyrG gene. pyrG+: the pyrG gene was constitutively expressed. pyrG+*: the pyrG gene was overexpressed. (D) Relative transcriptional levels of the pyrH gene. pyrH+: the pyrH gene was overexpressed. The ccpA gene was used as the internal control gene to normalize the results. Results are the average of three replications with error bars indicating standard error from the mean.
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Fig3: Relative transcriptional levels of mRNA in the recombinant strains. The mRNA expression levels of WT strain is regarded as 1(blank). The relative mRNA expression levels of recombinant strains (filled) are compared with WT strain. (A) Relative transcriptional levels of the pyrR gene. ΔpyrR: the pyrR gene was deleted. (B) Relative transcriptional levels of the prs gene. prs+: the prs gene was overexpressed. (C) Relative transcriptional levels of the pyrG gene. pyrG+: the pyrG gene was constitutively expressed. pyrG+*: the pyrG gene was overexpressed. (D) Relative transcriptional levels of the pyrH gene. pyrH+: the pyrH gene was overexpressed. The ccpA gene was used as the internal control gene to normalize the results. Results are the average of three replications with error bars indicating standard error from the mean.

Mentions: As described before, the transcription regulation mechanism of the pyr operon restricted the over-synthesis of UMP and its derivatives. We deleted 738 bp coding sequences of the pyrR gene in the strain TD02 and constructed the recombinant B. subtilis TD12 (Additional file 1: Figure S3). The pyr operon mRNA transcription level in the ΔpyrR strain TD12 was compared with parent strain TD02 through RT-qPCR analysis. We chose the sequences which lay in the middle of the pyr operon as the detective point. The transcript abundance increased 6.28-fold in TD12 (Figure 3), which indicated that the pyr operon was deregulated in ΔpyrR strain. The shake-flask culture experiments showed that the strain TD12 could accumulate 200.9 ± 8.3 mg/L cytidine, 14.9 ± 0.8 mg/L uridine and 960.1 ± 39.1 mg/L uracil, respectively (Figure 2). The deregulation of the pyr operon expression could significantly increase the accumulation of both cytidine and uracil, and have little effect on the accumulation of uridine in the medium.Figure 3


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)

Relative transcriptional levels of mRNA in the recombinant strains. The mRNA expression levels of WT strain is regarded as 1(blank). The relative mRNA expression levels of recombinant strains (filled) are compared with WT strain. (A) Relative transcriptional levels of the pyrR gene. ΔpyrR: the pyrR gene was deleted. (B) Relative transcriptional levels of the prs gene. prs+: the prs gene was overexpressed. (C) Relative transcriptional levels of the pyrG gene. pyrG+: the pyrG gene was constitutively expressed. pyrG+*: the pyrG gene was overexpressed. (D) Relative transcriptional levels of the pyrH gene. pyrH+: the pyrH gene was overexpressed. The ccpA gene was used as the internal control gene to normalize the results. Results are the average of three replications with error bars indicating standard error from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Relative transcriptional levels of mRNA in the recombinant strains. The mRNA expression levels of WT strain is regarded as 1(blank). The relative mRNA expression levels of recombinant strains (filled) are compared with WT strain. (A) Relative transcriptional levels of the pyrR gene. ΔpyrR: the pyrR gene was deleted. (B) Relative transcriptional levels of the prs gene. prs+: the prs gene was overexpressed. (C) Relative transcriptional levels of the pyrG gene. pyrG+: the pyrG gene was constitutively expressed. pyrG+*: the pyrG gene was overexpressed. (D) Relative transcriptional levels of the pyrH gene. pyrH+: the pyrH gene was overexpressed. The ccpA gene was used as the internal control gene to normalize the results. Results are the average of three replications with error bars indicating standard error from the mean.
Mentions: As described before, the transcription regulation mechanism of the pyr operon restricted the over-synthesis of UMP and its derivatives. We deleted 738 bp coding sequences of the pyrR gene in the strain TD02 and constructed the recombinant B. subtilis TD12 (Additional file 1: Figure S3). The pyr operon mRNA transcription level in the ΔpyrR strain TD12 was compared with parent strain TD02 through RT-qPCR analysis. We chose the sequences which lay in the middle of the pyr operon as the detective point. The transcript abundance increased 6.28-fold in TD12 (Figure 3), which indicated that the pyr operon was deregulated in ΔpyrR strain. The shake-flask culture experiments showed that the strain TD12 could accumulate 200.9 ± 8.3 mg/L cytidine, 14.9 ± 0.8 mg/L uridine and 960.1 ± 39.1 mg/L uracil, respectively (Figure 2). The deregulation of the pyr operon expression could significantly increase the accumulation of both cytidine and uracil, and have little effect on the accumulation of uridine in the medium.Figure 3

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