Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system / 生物工程学报

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.

Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production / 生物工程学报

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.

Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer / 上海交通大学学报（医学版）

Objective·To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro.Methods·The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract,and the PPK2 phylogenetic tree was constructed.The binding affinity of the PPK2 aptamer to H37Rv,BCG,Mycobacterium smegmatis,Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA).The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis.The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method.H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed.H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d,absorbance at 600 nm was measured by microplate reader.The effect of PPK2 aptamer on the growth of H37Rv was observed.Results·PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract,and it was relatively close to Pseudomonas aeruginosa.The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv.Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h.The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L.The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth.Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing trend with the increase of PPK2 aptamer concentration,which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth.Conclusion·PPK2 aptamer has good antibacterial activity against H37Rv in vitro.

Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer / 上海交通大学学报（医学版）

Objective: To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro. Methods: The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract, and the PPK2 phylogenetic tree was constructed. The binding affinity of the PPK2 aptamer to H37Rv, BCG, Mycobacterium smegmatis, Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA). The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis. The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method. H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed. H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d, absorbance at 600 nm was measured by microplate reader. The effect of PPK2 aptamer on the growth of H37Rv was observed. Results: PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract, and it was relatively close to Pseudomonas aeruginosa. The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv. Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h. The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L. The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth. Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing trend with the increase of PPK2 aptamer concentration, which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth. Conclusion: PPK2 aptamer has good antibacterial activity against H37Rv in vitro.

Polyphosphate kinase 1-coding gene (ppk1) is involved in the oxidative stress resistance in uropathogenic Escherichia coli by modulating the expression of katG and katE genes / 中华微生物学和免疫学杂志

Objective To investigate the role and the mechanism of ppk1 gene (coding for polyphosphate kinase 1) in oxidative stress resistance in uropathogenic Escherichia coli (UPEC).MethodsMutant strains with ppk1-deletion (△pk1) and complemented strains (△pk1-C) were constructed based on the UPEC strain CFT073.A comparative analysis was conducted to analyze survival rates of CFT073, △pk1 and △pk1-C strains at different time points while they were under oxidative stress.Differences in protein expression between CFT073 and △pk1 strains were analyzed using mass spectrometric analysis.Differences between CFT073 and △pk1 strains in expression of katG and katE genes were analyzed using real-time quantitative RT-PCR.Results The survival rate of △pk1 strains was lower than that of CFT073 strains at every time point, while the survival rate of △pk1-C strains was basically the same as that of CFT073 strains.Gel image analysis and mass spectrometric analysis revealed that six proteins were down-regulated and one was up-regulated in △pk1 strains as compared with those in CFT073 strains.Expression of the catalase-coding genes katG and katE in △pk1 strains were respectively (20.5±8.2)% and (20.9±6.9)% of those in CFT073 strains (P<0.05).Conclusion The ppk1 gene plays an important role in oxidative stress resistance in UPEC by modulating the expression of catalase-coding genes katG and katE.

Construction of recombinant strains co-expressing PPK and GMAS for the synthesis of L-theanine / 生物工程学报

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence. FINDINGS: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin. CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

Construction of uropathogenic Escherichia coli strain with ppk1 gene deletion and study on its biolog-ical properties / 中华微生物学和免疫学杂志

Objective To construct a Polyphosphate kinase 1 ( ppk1) gene deletion mutant of uro-pathogenic Escherichia coli (E.coli) CFT073, and to explore the biological properties of the mutant strain . Methods The ppk1 gene deletion strain (△pk1) was constructed based on CFT073 E.coli strain by usingλRed homologous recombination technology .A comparison analysis was conducted on adhesive and invasive abilities between CFT073 wild type strain and △pk1 strain in in vitro model of human bladder cancer epithe-lial cell 5637 .Crystal violet staining method was used to evaluate the influences of ppk1 gene deletion on biofilm formation.Results The CFT073 ppk1 deletion mutant strain was successfully constructed .Com-pared with the wild type strain ,△pk1 strain showed impaired adhesive and invasive abilities to 5637 cells. Moreover , the absorbance values of crystal violet at 570 nm at each time point of the mutant strain group were also lower than those of the wild-type strain group .Conclusion The ppk1 gene deletion mutant of uro-pathogenic E.coli CFT073 could be successfully constructed by Red homologous recombination technology and its biological properties indicates that ppk1 gene plays an important role in the pathogenesis of uropatho-genic E.coli infection through regulating the abilities of adhesion , invasion and biofilm formation .

Development and evaluation of protective capacity of Salmonella Enteritidis polyphosphate kinase-deleted and temperature-sensitive mutant / 대한수의학회지

This study was focusing on evaluating the protection of polyphosphate kinase (ppk) deleted and/or temperature-sensitive (ts) Salmonella Enteritidis (SE) as an attenuated vaccine in chickens. We constructed SEppk, SEts and SEppk::ts mutants and screened those mutants by growth capability in vitro, protection study in mice model and antibody response in chickens. Among the mutants, SEppk::ts-3 was selected because it showed higher growth capability, good protection against highly virulent SE in mice model, and good antibody response in chickens. SEppk::ts-3 also showed good protection against highly virulent SE isolate because it decreased colonization of virulent SE challenge strain in spleen, liver and cecum compared with the non-vaccinated control. The SEppk::ts-3 mutant showed cross-protection against S. Gallinarum (SG) challenge although the its cross-protection rate was a little lower than that of SG9R, a commercial vaccine against SG infection. To use for live attenuated vaccine in chickens, it should further be characterized.