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

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.

Figure 1

The biosynthetic pathway of pyrimidine nucleotide in B. subtilis.

Figure 2

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).

Figure 3

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.

Figure 4

Nucleotide sequence of P_SB expression cassette. The sequence of the nontemplate strand is shown. The −10 and −35 regions of the promoter, Shine–Dalgarno sequence (SD) and initiation codon are bold and underlined. The RNA-stabilizing elements are shown in italics.

Figure 5

The biomass of recombinant strains in 72 h fermentation. Data obtained are the result of three independent fermentations.

Figure 6

Nucleotide sequence of P_AE expression cassette. The sequence of the nontemplate strand is shown. The −10 and −35 regions of the promoter, transcription start site (+1), Shine–Dalgarno sequence (SD) and initiation codon are bold and underlined. The RNA-stabilizing elements are shown in italics.