Metabolic and genetic factors affecting the productivity of pyrimidine nucleoside in Bacillus subtilis
© Zhu et al.; licensee BioMed Central. 2015
Received: 20 November 2014
Accepted: 31 March 2015
Published: 15 April 2015
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.
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.
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.
KeywordsBacillus subtilis Cytidine Uridine Gene deletion Gene expression Pyrimidine nucleotide biosynthesis
In B. subtilis, L-aspartate is the precursor of both amino acids (lysine, methionine, threonine, and isoleucine) and pyrimidine nucleotide biosynthesis (Figure 1). The deficiency of homoserine dehydrogenase (encoded by the hom gene) can prevent the aspartate entering to the methionine and threonine biosynthesis and improve the supplement to the de novo biosynthesis of UMP . Phosphoribosyl pyrophosphate (PRPP) is another important precursor of pyrimidine nucleotide biosynthesis (Figure 1). Phosphoribosyl pyrophosphate synthetase (PRS) is encoded by the prs gene of the gcaD-prs-ctc operon whose transcription regulation mechanism has been unknown yet . Improving the expression level of the prs gene can significantly increase the intracellular pool of PRPP [7,8].
In B. subtilis, UMP kinase, which is encoded by the pyrH gene, catalyzes the phosphorylation of UMP by ATP to yield UDP and ADP (Figure 1). The UMP kinase activity is regulated allosterically by GTP (activator) and UTP (inhibitor) [9,10]. The pyrG gene encodes CTP synthetase which aminates UTP to form CTP (Figure 1). The transcription of the pyrG gene is tightly regulated by a CTP-sensitive reiterative transcription attenuation control mechanism [11-13]. The inherent regulation of the pyrG gene transcription and CTP synthetase activity limit the excessive synthesis of CTP in B. subtilis.
Catalyzed by non-specific 5′-phosphatase, the excessive intracellular CMP and UMP can be dephosphorylated to form cytidine and uridine, respectively. The cytidine can be further deaminized to form uridine by cytidine deaminase (encoded by the cdd gene) (Figure 1) . In the dra-nupC-pdp operon, the pdp gene encodes pyrimidine nucleoside phosphorylase which catalyzes the degradation of uridine to form uracil and D-ribose-1-phosphate . Then, uracil will be secreted out of the cell. Therefore, the terminal metabolites of pyrimidine nucleotide are generally uracil instead of cytidine and uridine in the wild-type B. subtilis. The dra gene encodes deoxyriboaldolase and the nupC gene encodes the transporter responsible for pyrimidine nucleoside uptake. The expression of the dra-nupC-pdp operon is repressed by glucose and induced by deoxyribonucleosides and deoxyribose [16,17]. When the carbon source is poor, by the physiological function of the dra-nupC-pdp operon, the pyrimidine nucleoside accumulated in the medium could be recycled as carbon source. Obviously, the physiological functions of nupC and pdp genes make no contribution to the accumulation of pyrimidine nucleoside in the medium.
B. subtilis strains with defect and pyrimidine analogue resistant could accumulate large amounts of cytidine or uridine [18-21]. Nevertheless, the exact genetic and metabolic mechanisms resulting in pyrimidine nucleoside over-production have not been fully identified and confirmed.
In this study, by using genetic manipulation method, we modified some key genes and operons related to the pyrimidine nucleotide biosynthesis in B. subtilis 168 and investigated the influence of these modifications on the production of pyrimidine nucleoside compounds.
Deletion of the cdd and hom genes
Deregulation of the pyr operon
Overexpression of the prs gene
PRPP is not only a precursor for UMP de novo biosynthesis but also locates in the crossing point in multiple metabolic pathways. To illustrate the impact of intracellular PRPP level on the biosynthesis of UMP and its derivatives, we overexpressed the prs gene in TD12 and constructed recombinant strain B. subtilis TD13. The RT-qPCR analyses showed that a 72.86-fold improvement of the prs mRNA transcription level occurred in recombinant TD13 compared with parental strain TD12, which indicated that the prs gene was successful overexpressed (Figure 3). The flask culture revealed that the recombinant strain TD13 accumulated 247.2 ± 9.6 mg/L cytidine, 440.7 ± 17.1 mg/L uridine and 935.7 ± 31.9 mg/L uricil in medium, respectively (Figure 2). Compared with strain TD12, the accumulation of cytidine and uracil of strain TD13 slightly rose or dropped, but the accumulation of uridine significantly increased by 25.9-fold. These results demonstrated that pyrimidine nucleotide precursor can be further increased by overexpressing the prs gene, and intracellular PRPP level was an important rate-limiting factor in biosynthesis of UMP and its derivatives. In other words, the improvement of PRPP supplement could increase the synthesis of pyrimidine nucleoside which was mainly reflected in the improvement of uridine.
Overexpression of the pyrG gene
Overexpression of the pyrH gene
Among the reactions from UMP to cytidine, UMP kinase (encoded by the pyrH gene) might serve as a rate-limiting factor. In order to confirm this, we overexpressed the pyrH gene in strain TD232 and obtained the recombinant B. subtilis TD33. The RT-qPCR analyses showed that the pyrH mRNA level of TD33 was about 3.75-fold higher than that of strain TD232 of which the pyrH gene was wild-type (Figure 3). The shake flask fermentation experiments showed that the recombinant TD33 accumulated 1244.2 ± 53.9 mg/L cytidine, 558.6 ± 24.2 mg/L uridine and 1696.7 ± 39.7 mg/L uracil in the culture broth and increased by 40%, 14% and 7.5%, respectively, compared with strain TD232 (Figure 2). The production of cytidine, uridine and uracil all increased with the improvement of UMP kinase express level. These results proved that UMP kinase express level was also a rate-determining factor of pyrimidine nucleoside compounds production.
Disruption of the nupC-pdp gene
Pyrimidine and pyrimidine nucleoside produced by modifying different genes of the pyrimidine nucleotide biosynthesis pathway in B. subtilis after 72 h fermentation
200.9 ± 8.3
14.9 ± 0.8
960.1 ± 39.1
260.3 ± 11.3
1187.5 ± 49.9
508.2 ± 18.5
247.2 ± 9.6
440.7 ± 17.1
935.7 ± 31.9
326.9 ± 7.0
1571.4 ± 38.9
16.9 ± 0.1
1244.2 ± 53.9
558.6 ± 24.2
1696.7 ± 39.6
1423.0 ± 47.5
1684.6 ± 62.4
14.2 ± 0.2
Together with the inactivation of the cdd, hom and pyrR genes, the deletion of nupC-pdp gene led to a slight increase of the total amount of pyrimidine compounds (i.e., cytidine, uridine, and uracil) accumulated in the culture. However, together with the inactivation of the cdd, hom and pyrR genes and the overexpression of the prs gene, the deletion of the nupC-pdp gene led to a modest decrease of the total amount of pyrimidine compounds in the culture. Nevertheless, if the pyrH and pyrG genes were both overexpressed subsequently, the total amount of pyrimidine compounds in the culture would be doubled to about 22 mmol/L (Table 1). These results revealed that the overexpression of the pyrH and pyrG genes caused a metabolic flux enlargement from UMP to cytidine, thereby, reducing the intracellular UMP level which may relieve feedback inhibition for UMP to carbamyl phosphate synthetase and lead to more UMP synthesis.
The growth of recombinant strains
As already shown in the “introduction”, the pyrimidine nucleotide biosynthesis of B. subtilis was strictly regulated so that no excess pyrimidine nucleoside would be synthesized and secreted to the medium. In order to well illuminate the rate-determining factors affecting pyrimidine nucleoside excess synthesis, we chose genetic engineering as a desired strategy.
The inactivation of the cdd gene abolished the reaction from cytidine to uridine and resulted in the accumulation of cytidine in the medium, indicating that the deficiency of the cdd gene is a key factor of the accumulation of cytidine. The result coincided with the experimental findings of previous studies .
Based on the hom and cdd genes deficiency, the deregulation of the pyr operon by deleting the pyrR gene doubled the yield of both cytidine and uracil, while mutagenesis was the mere prevailing method to achieve the same genetic effect in the past . However, though one determining factor of UMP biosynthesis have been revealed with deregulation of the pyr operon, the carbamoyl phosphate synthetase (encoded by the pyrAA/pyrAB gene), which catalyzes the first reaction of UMP de novo biosynthesis, is still subjected to feedback inhibition by UMP [23,24]. Hence, the excess synthesis of pyrimidine nucleoside compounds is still considered to be limited.
The overexpression of the prs gene resulted in an increase of uridine yield by 25.9-fold in this work. Analogous experimental results also existed in the study of biosynthesis of purine nucleoside and its derivatives in B. subtilis [8,25]. Thus, PRPP pool level is one important restrictive factor affecting the overproduction of nucleotide and its derivatives. However, PRS is an allosteric enzyme and its activity is intensively feedback inhibited by purine nucleotide . By using site-directed mutation to release the feedback inhibition of phosphoribosyl pyrophosphate synthetase (PRS), satisfactory results may be obtained.
The overexpression of the pyrG and pyrH genes resulted in a significantly increased amount of cytidine in the medium, proving that the activity of both UMP kinase and CTP synthetase were rate-determining factors for cytidine over-synthesis. Since both UMP kinase and CTP synthetase are allosteric enzymes, it is believed that by releasing their feedback inhibition, the cytidine production could be further improved. The overexpression of the pyrG and pyrH genes also led to an increase in the total pyrimidine compounds accumulation in the medium. We interpret that the augmented metabolic flux from UMP to CTP could reduce the intracellular UMP level and thereby release the feedback inhibition on UMP biosynthesis, especially the activity of carbamyl phosphate synthetase.
The pyrimidine nucleoside phosphorylase (encoded by the pdp gene) involves in the reaction from uridine to uracil and the loss of the pdp gene should make cell accumulate only nucleosides and no uracil in theory [27,28]. However, according to the experimental results, by deleting the nupC-pdp gene, we obtained that the uracil accumulation reduced by 99% rather than 100%. The small amount of uracil remaining in the medium likely resulted from some unspecific reactions. Since the decrease of uracil yield was always accompanied with a remarkable increase of uridine yield, the uracil accumulated in the medium was mainly derived from uridine or UMP. Under the precondition of the pdp gene deficiency, the amount of residual uracil in the medium seems to be negatively correlated with the amount of the available PRPP in the cell (TD12np). Hence, the unspecific reactions which result in the residual uracil are likely subject to the level of intercellular PRPP pool.
We deleted/overexpressed the genes which were closely related to pyrimidine nucleoside biosynthesis by using genetic manipulation method in B. subtilis168 and constructed a series of recombinant strains. The results of shaking flask fermentation demonstrated that the deregulation of pyr operon, the overexpression of the prs, pyrG and pyrH genes, and the deletion of the nupC-pdp gene all facilitated the over-synthesis of pyrimidine nucleoside compounds. The production of uridine and cytidine were up to 1684.6 mg/L and 1423 mg/L in the result strain TD33np, respectively. (i) The UMP synthesis operon (pyr operon) and the PRPP synthesis (encoded by the prs gene) are both rate-determining factors for the UMP biosynthesis. If the feedback inhibitions of PRPP synthetase and carbamoyl phosphate synthetaserelease are released, the production of pyrimidine nucleoside compounds may be improved further. (ii) The overexpression of the pyrH and pyrG genes can improve the proportion of cytidine in the pyrimidine nucleoside products, while reduce the proportion of uridine. If the feedback inhibition of the CTP synthetase and UMP kinase are released, the proportion of cytidine in the pyrimidine nucleoside products may be improved further. (iii) The pyrimidine nucleoside phosphorylase (encoded by the pdp gene) activity is of close correlation with the accumulation of uracil in the medium, and the deletion of the nupC-pdp gene can reduce the accumulation of uracil to a very low level (1%) in the medium. (iv) The cdd gene is a key factor of the accumulation of cytidine, and if cytidine deaminase activity is restored, the proportion of uridine in the accumulated pyrimidine compounds will increase sharply.
Materials and methods
The bacterial strains and general culture conditions
B.subtilis 168 (BGSC 1A1)
B.subtilis 168 N
trpC2, ΔaraR::neo R
trpC2, ΔaraR::neo R ,Δcdd
trpC2, ΔaraR::neo R , Δcdd, Δhom
trpC2,ΔaraR::neo R , Δcdd, Δhom, ΔpyrR
trpC2,ΔaraR::neo R , Δcdd, Δhom, ΔpyrR, ΔnupC-pdp
trpC2,ΔaraR::neo R , Δcdd, Δhom,ΔpyrR, ΔxylR::prs
trpC2,ΔaraR::neo R , Δcdd, Δhom,ΔpyrR, ΔxylR::prs, ΔnupC-pdp
trpC2,ΔaraR::neo R , Δcdd, Δhom, ΔpyrR, ΔxylR::prs, pyrG +
trpC2,ΔaraR::neo R , Δcdd, Δhom, ΔpyrR,ΔxylR::prs, pyrG +*
trpC2,ΔaraR::neo R , Δcdd, Δhom, ΔpyrR, ΔxylR::prs,pyrG +* ,pyrH +
trpC2,ΔaraR::neo R , Δcdd, Δhom, ΔpyrR,ΔxylR::prs,pyrG +* ,pyrH + , ΔnupC-pdp
DNA manipulation techniques and PCR
The isolation and manipulation of DNA were conducted according to standard procedures . All chromosomal DNA were extracted from B. subtilis and isolated by the protocol of Sangon Biotech (Shanghai, China). PCR was performed with DNA Ploymerase HiFi or Taq DNA polymerase (TransGen, Beijing, China) in a DNA thermal cycler (DNAEngine, BIO-RED, Hercules, CA, USA) through the procedure recommended by the manufacturer. Overlapped extension PCR (SOE-PCR) was carried out as decribed [30,31]. PCR products were purified with a PCR Purification kit (Biomed, Beijing, China) and analyzed by electrophoresis in 1% (w/v) agarose gels.
Transformation and transformants’ seletion
Transformation of B. subtilis was performed by using competent cells as described by Anagnostopoulos and Spizizen . Competent transformation used liner DNA frangments. The transformants seletion relied on the method described by Liu et al. [33,34].
Primers and synthetized fragments
Oligonucleotides for pyrR gene deletion
Sequence 5'- 3'
Oligonucleotides for pyrH gene overexpression
Sequence 5'- 3'
Oligonucleotides for prs gene overexpression
Sequence 5'- 3'
Oligonucleotides for hom gene deletion
Sequence 5'- 3'
Oligonucleotides for nupC-pdp gene deletion
Sequence 5'- 3'
Oligonucleotides for cdd gene deletion
Sequence 5'- 3'
Oligonucleotides for pyrG gene constitutive expression and overexpression
Sequence 5'- 3'
A loop of cells grown on an agar plate of LB medium was inoculated into a 250 mL flask containing 30 mL of stock culture medium (2% glucose, 2% soybean meal hydrolysate, 1% yeast extract, 0.25% NaCl, 0.1% MgSO4.7H2O, 0.1% KH2PO4, 0.5% sodiumglutamate, pH = 7.0) and was then cultured for 16 h at 37°C, with shaking at 220 rpm. 1.5 mL of the culture was transferred to a 250 mL flask containing 30 mL of fermentation medium (8% glucose, 3% soybean meal hydrolysate, 2% cornsteepliquor, 1.5% yeast extract, 0.25% NaCl, 0.8% MgSO4.7H2O, 0.25% KH2PO4, 1.5% (NH4)2SO4, 1.5% sodiumglutamate, pH = 7.0) and was then cultured for 72 h at 37°C, with shaking at 220 rpm. Samples were drawn at various time-points during the fermentation and were analyzed for cell growth (OD600) and pyrimidine compounds content.
Fermentation compounds analysis
The qualitative analysis of pyrimidine nucleoside and pyrimidine compounds in the medium was conducted by massspectrograph (Q-Exactive, Thermo Scientific, Waltham, MA, USA).The quantitative analysis of cytidine, uridine and uracil were conducted by HPLC (Waters 2695, Waters, Milford, MA, USA) with an HYPERSIL ODS C18 column (Thermo Scientific, Waltham, MA, USA). Separation was performed at 40°C with 0.05 M KH2PO4 at a flowrate of 1.5 mL min−1. The detective wavelength was 270 nm.
Quantitative real-time reverse transcription (RT)-PCR analysis
Total RNA of B. subtilis was extracted with RNAprep pure Cell/Bacteria Kit (TIANGEN, Beijing, China) as recommended by the supplier and was reverse-transcribed in cDNA using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The Real-time PCR was carried out by LightCycler 480 (Roche Diagnostics GmbH, Mannheim, Germany) using SYBR Green Ι Master (Roche Diagnostics GmbH, Mannheim, Germany). The ccpA gene was served as internal control of Real-time PCR. The quantification cycle (Cq) was determined according to the second derivative maximum method using the LightCycler software 4.1 (Roche Diagnostics GmbH, Mannheim, Germany). The relative expression ratio (RE) was calculated according to Pfaffl .
We gratefully acknowledge the generous support of Lei Guo and Dr. Xi-xian Xie from Tianjin University of Science and Technology in fermentation experiments. We also appreciate Thermo Fisher Scientific for its important contributions (Shanghai, China) to qualitative analyses of fermentation products with Mass Spectrometry.
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