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L-Asparagine, a crucial amino acid widely used in both food and medicine, presents pollution-related and side reaction challenges when prepared using chemical synthesis method. Although biotransformation methods offer significant advantages such as high efficiency and mild reaction conditions, they also entail increased costs due to the need for ATP supplementation. This study aimed to address the challenges associated with biopreparation of L-asparagine. Firstly, the functionality and characteristics of recombinant L-asparagine synthetase enzymes derived from Escherichia coli and Lactobacillus salivarius were evaluated to determine their practical applicability. Subsequently, recombinant expression of polyphosphate kinase from Erysipelotrichaceae bacterium was conducted. A reaction system for L-asparagine synthesis was established using a dual enzyme-coupled conversion approach. Under the optimal reaction conditions, a maximum yield of 11.67 g/L of L-asparagine was achieved, with an 88.43% conversion rate, representing a 5.03-fold increase compared to the initial conversion conditions. Notably, the initial addition of ATP was reduced to only 5.66% of the theoretical demand, indicating the effectiveness of our ATP regeneration system. These findings highlight the potential of our approach in enhancing the efficiency of L-asparagine preparation, offering promising prospects for the food and medical industries. [ABSTRACT FROM AUTHOR]

The linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In many Lactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1 and ppx2) each having different domain compositions, with the gene order ppx1-ppk-ppx2. However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans. Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis in Lc. paracasei and have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis. [ABSTRACT FROM AUTHOR]

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum (SlPPK2). After directed evolution and computation-supported design, we found that SlPPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of SlPPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effective ly avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this study, a dual-bacterial coupled fermentation system containing nicotinamide nucleoside kinase (NRK) and polyphosphatase (PPK) was constructed, and the application of PPK-based ATP regeneration system in NMN production was achieved. First, engineering strains expressing NRK1 and NRK2 were constructed, and the highly active Escherichia coli BL21 (DE3)-pET28a-NRK1 was selected, with NMN yield and productivity of 5.17 g/L and 77.4%, respectively. Then, the induced expression conditions of NRK1 were optimized, and a low temperature of 16 ℃, an isopropyl-β-D-thiogalactopyranoside (IPTG) concentration of 0.7 mmol/L, an inoculation amount of 3% and an induction duration of 22 h were found to be optimal the soluble expression of NRK1 protein. The optimal synthesis conditions of NMN by E. coli BL21 (DE3)-pET28a-NRK1 were explored. It was found that after 12 h culture at 18 ℃ at an initial cell concentration of 100 g/L and a ratio of ATP to NR of 1:1.5, the highest yield of NMN of 5.73 g/L was obtained with a productivity of 85.78%. Finally, the optimal conditions that provided maximal NMN production (11.81 g/L) by coupled fermentation with E. coli BL21 (DE3) pET28a-PPK and E. coli BL21 (DE3)-pET28a-NRK1 were determined as 1:3.5, 1:2 and 16 h for ATP to NR ratio, initial cell concentration and fermentation time, respectively. The high-density dual-bacterial coupled fermentation strategy established in this study opens up a new pathway for high-efficiency, low-cost and large-scale production of NMN.

Abstract ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

d-Tagatose is a rare sugar with low calories, and is extensively used in food, beverage, and drug additives. In this study, an in vitro multienzyme cascade route for d-tagatose synthesis from sucrose (MCTS) was designed, which contains five enzymes (sucrose phosphorylase, fructokinase, d-fructose 6-phosphate 4-epimerase, d-tagatose 6-phosphate phosphatase, and polyphosphate kinase). The whole MCTS route comprised a sucrose phosphorylation reaction, and a phosphorylation–dephosphorylation reaction coupled with an ATP regeneration system. After optimization, the conversion of d-tagatose from 10 mM sucrose reached 82.3%. At an elevated sucrose concentration of 50 mM, 72.4% of d-tagatose conversion and 0.27 g·L–1·h−1 of space–time yield were obtained. Furthermore, ADP consumption decreased to 1% of the sucrose concentration after introducing the ATP regeneration system. The MCTS strategy is an efficient and cost-effective approach for d-tagatose production. [ABSTRACT FROM AUTHOR]

Adenosine 5'-triphosphate (ATP) is a substrate for the biological synthesis of various valuable compounds, such as pharmaceuticals and agrochemicals. To make these biocatalytic processes more economically valuable, several ATP regeneration systems comprising enzymes and phosphate donors were developed. Due to their ability to synthesize ATP from both 5'-diphosphate and adenosine 5'-monophosphate, polyphosphate kinase 2 class III (PPK2-III) enzymes such as Cytophaga hutchinsonii PPK (ChPPK) have attracted considerable attention in the construction of ATP regeneration systems. Furthermore, PPK2-III enzymes employ inorganic polyphosphate, which is a low-cost and stable substrate, as the phosphate donor. To facilitate the application of the PPK2-III ATP regeneration system in biocatalytic processes depending on ATP, a semirational strategy coupled with a high-throughput screening assay based on a fluorescent sensor was performed to engineer ChPPK. Saturation mutagenesis was performed on 16 critical residues of ChPPK. After screening approximately 4,800 colonies, nine single amino acid variants exhibited more than threefold increased activities than wild-type ChPPK. Beneficial mutations were subsequently recombined, resulting in a quadruple variant (ChPPK/A79G/S106C/I108F/L285P), compared with wild-type ChPPK, displaying 18.8-fold enhanced activity and better stability at an elevated temperature and under acidic conditions. The introduction of this variant ChPPK/A79G/S106C/I108F/L285P in the biological synthesis of nicotinamide mononucleotide catalyzed by phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyltransferase increased the yield by approximately threefold compared with wild-type ChPPK. Therefore, the ATP regeneration system described may have implications for bioconversions requiring ATP. IMPORTANCE The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK (ChPPK). However, the wild-type ChPPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of ChPPK, we employed a semi-rational design approach to obtain the variant ChPPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant ChPPK/A79G/S106C/I108F/ L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes. [ABSTRACT FROM AUTHOR]

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non‐canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS‐CoV‐2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7‐deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency. [ABSTRACT FROM AUTHOR]

ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably. [ABSTRACT FROM AUTHOR]

The multi-enzyme coupling reaction system has become a promising biomanufacturing platform for biochemical production. Tyr is an essential amino acid, but the limited solubility restricts its use. Tyrosyl dipeptide has been paid more attention due to its higher solubility. In this study, an efficient enzymatic cascade of Ala-Tyr synthesis was developed by a L-amino acid ligase together with polyphosphate kinase (PPK). Two L-amino acid ligases from Bacillus subtilis and Bacillus pumilus were selected and applied for Ala-Tyr synthesis. The L-amino acid ligase from B. subtilis (Bs) was selected and coupled with the PPK from Sulfurovum lithotrophicum (PPKSL) for regenerating ATP to produce Ala-Tyr in one pot. In the optimization system, 40.1 mM Ala-Tyr was produced within 3 h due to efficient ATP regeneration with hexametaphosphate (PolyP(6)) as the phosphate donor. The molar yield was 0.89 mol/mol based on the substrates added, while the productivity of Ala-Tyr achieved 13.4 mM/h, which were the highest yield and productivity ever reported about Ala-Tyr synthesis with L-amino acid ligase. [ABSTRACT FROM AUTHOR]

Abstract Background ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered. Results In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2B cg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains. Conclusions In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.

Background: ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered. Results: In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2Bcg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains. Conclusions: In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites. [ABSTRACT FROM AUTHOR]

Polyphosphate kinase 1 (Ppk1) generates polyphosphates (polyPs) by catalyzing phosphate transfer from ATP. In the presence of ATP, Myxococcus xanthus Ppk1 showed the highest activity with polyP60–70 but also showed high activity with orthophosphate and pyrophosphate. Ppk1 synthesizes long-chain polyPs with >1 000 phosphate residues from orthophosphate or pyrophosphate present in high concentrations, suggesting that in M. xanthus , Ppk1 uses intracellular ortho/pyrophosphate as an initial primer for polyP production. During M. xanthus starvation-induced development, the specific activity of Ppk1 peaked at 12 h (300–800 nmol/min/mg) and then gradually decreased. The polyP concentration was highest during mound formation (45 nmol phosphate/mg protein); then, the level of long-chain polyPs decreased and that of short-chain polyPs increased during fruiting body and spore formation. Myxococcus xanthus expresses two exopolyphosphatases, Ppx1 and Ppx2, which mainly degrade short- and long-chain polyPs, respectively, both of which were highest in vegetative cells and were detected during starvation, which may account for the degradation of polyPs. Thus, polyPs synthesized by Ppk1 early in starvation-induced development could be degraded by exopolyphosphatases and may also be used as substrates by polyP:AMP phosphotransferases and polyphosphate/ATP-NAD kinases to generate ADP and NADP+, respectively. [ABSTRACT FROM AUTHOR]

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high-performance liquid chromatography tandem mass spectrometry. Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. Our study shows that PPKs are promiscuous enzymes in vitro that could be involved in the formation of a variety of phosphorylated metabolites in vivo. [ABSTRACT FROM AUTHOR]

The most unique characteristic of Mycobacterium tuberculosis is persistence in the human host, and the biofilm formation is related to the persistance. Polyphosphate (polyP) kinase 1 (PPK1) is conserved in Mycobacteria and is responsible for polyP synthesis. polyP is a chain molecule linked by high-energy phosphate bonds, which is considered to play a very important role in bacterial persistence. However, the relationship of PPK1 and mycobacterial biofilm formation is still adequately unclear. In current study, ppk1-deficient mutant (MT), ppk1-complemented (CT) and wild-type strains of M. smegmatis mc2 155 were used to investigate the formation, morphology and ultramicrostructure of the biofilm and to analyze the lipid levels and susceptibility to vancomycin antibiotic. And then WT, MT and CT strains were used to infect macrophages and to analyze the expression levels of various inflammatory factors, respectively. We found that PPK1 was required for M. smegmatis polyP production in vivo and polyP deficiency not only attenuated the biofilm formation, but also altered the phenotype and ultramicrostructure of the biofilm and reduced the cell lipid composition (except for C16.1 and C17.1, most of the fatty acid components from C8–C24). Moreover, the ppk1-deficient mutant was also significantly more sensitive to vancomycin which targets the cell wall, and its ability to survive in macrophages was decreased, which was related to the change of the expression level of inflammatory factors in macrophage. This study demonstrates that the PPK1 can affect the biofilm structure through affecting the content of short chain fatty acid and promote intracellular survival of M. smegmatis by altering the expression of inflammatory factors. These findings establish a basis for investigating the role of PPK1 in the persistence of M. tuberculosis, and provide clues for treating latent infection of M. tuberculosis with PPK1 as a potential drug target.

β-nicotinamide mononucleotide (NMN) was a naturally occurring active derivative of B vitamins, which had the function of improving the mammalian fertility ability and intestinal function. Escherichia coli BW25113 was used as the expression host, and according to the protein sequence of nicotinamide ribokinase (NRK) in the NCBI database, Nrk genes from different sources were synthesized by homologous alignment analysis. Then, a recombinant strain NRK-Gs/BW with high efficiency in converting nicotinamide ribose (NR) to NMN was screened. 43 mmol • L-1 NMN could be synthesized in the reaction mixture containing 50 mmol • L-1 NR and 10 mmol • L-1 ATP. Polyphosphate kinase (PPK2) was further introduced to construct the ATP circulation system. The amount of ATP was reduced by 80%, and the AMP production in the reaction mixture was less than or equal to 3 mmol • L-1. Finally, the reaction mixture was optimized by factorial design. In the reaction mixture of 50 mmol • L-1 NR and 10 mmol • L-1 ATP, the yield of NMN was 49.96 mmol • L-1, with a molar yield of 99. 96%. By constructing the NMN engineering bacteria synthesized by NR containing the ATP circulation system, the molar ratio of ATP was greatly reduced, which laid a foundation for the subsequent production of NMN. [ABSTRACT FROM AUTHOR]

Zeolitic imidazole frameworks (ZIFs) with tunable pore sizes and large surface areas have been widely used as effective supports for enzyme/cell immobilization. In this study, a bi-enzyme system (BP@ZIF-8) was developed for the biosynthesis of Ala-Gln coupled with efficient ATP regeneration. BP@ZIF-8 was then successfully fabricated and characterized. Results showed that BP@ZIF-8 exhibited high thermostability at 50 °C and high pH stability in neutral and alkaline reaction buffers. However, BP@ZIF-8 displayed poor pH stability in acidic environments. In addition, it could maintain 58.9% of their initial activity after 7 continuous uses, demonstrating satisfactory reusability. When applied for the batch production of Ala-Gln, the yield of Ala-Gln reached to about 14.56 mM after 14 h, an increase of approximately 14.3%. These demonstrated that BP@ZIF-8 shows great potential for the practical biosynthesis of Ala-Gln. [ABSTRACT FROM AUTHOR]

Abstract Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract

Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1AT) is responsible for the formation of polyP granules, while the other (PPK2AT) is used for replenishing the NTP pools by using polyP as a phosphate donor to phosphorylate nucleoside diphosphates. Fusions of eYFP with PPK2AT or of the polyP granule-associated phosin PptA from Ralstonia eutropha always co-localized with polyP granules in A. tumefaciens and allowed the tracking of polyP granules in time-lapse microscopy experiments without the necessity to label the cells with the toxic dye DAPI. Fusions of PPK1AT with mCherry formed fluorescent signals often attached to, but not completely co-localizing with, polyP granules in wild-type cells. Time-lapse microscopy revealed that polyP granules in about one-third of a cell population migrated from the old pole to the new cell pole shortly before or during cell division. Many cells de novo formed a second (nonmigrating) polyP granule at the opposite cell pole before cell division was completed, resulting in two daughter cells each having a polyP granule at the old pole after septum formation. Migration of polyP granules was disordered in mitomycin C-treated or in PopZ-depleted cells, suggesting that polyP granules can associate with DNA or with other molecules that are segregated during the cell cycle. [ABSTRACT FROM AUTHOR]

• Virus-encoded polyphosphate kinases enhanced phosphorus removal in activated sludge • ppk genes were present in unclassified viral contigs of activated sludge • Viral PPKs contained presumed active sites and conserved residues • Experimental validation confirmed the roles of viral PPKs in phosphate removal While microbial phosphate removal in activated sludge (AS) systems has been extensively studied, the role of viruses in this process remains largely unexplored. In this study, we identified 149 viral auxiliary metabolic genes associated with phosphorus cycling from 2,510 viral contigs (VCs) derived from AS systems. Notably, polyphosphate kinase 1 (ppk1) and polyphosphate kinase 2 (ppk2) genes, which are primarily responsible for phosphate removal, were found in five unclassified VCs. These genes exhibited conserved protein structures and active catalytic sites, indicating a pivotal role of viruses in enhancing phosphorus removal. Phylogenetic analysis demonstrated a close relationship between viral ppk genes and their bacterial counterparts, suggesting the occurrence of horizontal gene transfer. Furthermore, experimental assays validated that viral ppk genes enhanced host phosphate removal capabilities. VCs carrying ppk genes were observed across diverse ecological and geographical contexts, suggesting their potential to bolster host functions in varied environmental and nutrient settings, spanning natural and engineered systems. These findings uncover a previously underappreciated mechanism by which viruses enhance phosphate removal in wastewater treatment plants. Overall, our study highlights the potential for leveraging virus-encoded genes to improve the efficiency of biological phosphorus removal processes, offering new insights into the microbial ecology of AS systems and the role of viruses in biogeochemical cycling. [Display omitted] [ABSTRACT FROM AUTHOR]

Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the in vitro multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.

Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. [ABSTRACT FROM AUTHOR]

Francisca Campos,1 Javiera A Álvarez,1 Javiera Ortiz-Severín,1 Macarena A Varas,1 Carlos F Lagos,2 Ricardo Cabrera,3 Sergio A Álvarez,4 Francisco P Chávez11Laboratorio de Microbiología de Sistemas, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile; 2Chemical Biology & Drug Discovery Laboratory, Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago de Chile, Chile; 3Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile; 4Laboratorio de Microbiología, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas Y Farmacéuticas, Universidad de Chile, Santiago de Chile, ChileAbstract: Inorganic polyphosphate (polyP) and its metabolic enzymes are important in several cellular processes related with virulence and antibiotic susceptibility. Accordingly, bacterial polyP synthesis has been proposed as a good target for designing novel antivirulence molecules as alternative to conventional antibiotics. In most pathogenic bacteria, polyphosphate kinase 1 (PPK1), in charge of polyP synthesis from ATP, is widely conserved. Current colorimetric and radioactive polyP synthesis enzymatic assays are not suitable for high-throughput screening of PPK1 inhibitors. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4ʹ-6-diamidino-2-phenylindole), a fluorescence assay was previously developed by using a purified recombinant PPK1 enzyme from Escherichia coli. In this work we have developed a suitable methodology for high-throughput measurement of E. coli PPK1 activity. This platform can be used for the screening putative antimicrobial molecules for related enteropathogenic bacteria.Keywords: antivirulence, antimicrobial, affinity purification, polyphosphate kinase, DAPI, fluorescence enzymatic activity

The article presents the astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses. Topics include the polyP kinases will critically assess of the underlying data on the multiple functions of polyP, and the most other functions of polyP are not relevant for survival of Ralstonia eutropha important beta-proteobacterial species.

The cell free system has been paid more attention due to its potential of facilitating efficient catalysis of multistep reactions. In this study, an efficient enzymatic cascade of glutathione (GSH) production was developed through the evolution of bifunctional glutathione synthetase (GshF), coupled with polyphosphate kinase (PPK). First, the stability and activity of GshF were enhanced by loop interchange and site‐directed mutagenesis. As a result, the GshF half‐value period increased 163.32‐fold, and its activity raised 18.16%. PPK from Jhaorihella thermophile (PPKJT) was characterized and used to regenerate ATP in the GSH synthesis, with hexametaphosphate (PolyP (6)) as the phosphate donor. After the process optimization, 99.92 mM GSH and 7.61 mM oxidized glutathione (GSSG) were produced within 2 hr. The molar yield was 0.96 mol/mol based on the amino acid added, while the productivities of GSH achieved 49.95 mM/hr, which were the highest yield and productivity ever reported about GSH synthesis. [ABSTRACT FROM AUTHOR]

Glutathione is a tri-peptide consisting of L-glutamic acid, L-cysteine and glycine, widely applied to the pharmaceutical, food, and cosmetic industries. Also, GSH has potential application in the bioremediation of soil and water. A novel ATP regulating strategy based on the ATP-sensing riboswitch ydaO and polyphosphate kinase was developed. Besides, GshF from Streptococcus thermophile was introduced into the Escherichia coli BL 21. The GSH titer of the strain AYPGS(harboring pRSFDuet-ydaO-ppk-gshF) could reach 145.32 mg L−1 after 18 h fermentation, which was three times higher than that of the control and 37% higher than that of the strain AGP(harboring pRSFDuet-ppk-gshF). The results showed that S. thermophiles-derived GshF was insensitive to feedback inhibition caused by GSH and could greatly increase the GSH production. It also proved that the ATP regulating strategy could dynamically control the ATP level and enhance GSH production. Our proposed ATP regulating strategy will be potentially applicable for other ATP-driven metabolites.

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosis. D. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis. Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data—in combination with in vivo results—suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification. Key points: •PPK1ATand PPK2AThave different substrate specificities, •PPK2ATis a subgroup 1 member of PPK2s, •PPK2ATcatalyzes the formation of polyphosphorylated nucleosides [ABSTRACT FROM AUTHOR]

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γ-Glutamyl compounds have unveiled their importance as active substances or precursors of pharmaceuticals. In this research, an approach for enzymatic synthesis of γ-glutamyl compounds was developed using γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays and polyphosphate kinase (PPK) from Corynebacterium glutamicum. GMAS and PPK were co-recombined in pETDuet-1 plasmid and co-expressed in E. coli BL21 (DE3), and the enzymatic properties of GMAS and PPK were investigated, respectively. Under the catalysis of the co-expression system, l-theanine was synthesized with 89.8% conversion when the substrate molar ratio of sodium glutamate and ethylamine (1:1.4) and only 2 mM ATP were used. A total of 14 γ-glutamyl compounds were synthesized by this one-pot method and purified by cation exchange resin and isoelectric point crystallization with a yield range from 22.3 to 72.7%. This study provided an efficient approach for the synthesis of γ-glutamyl compounds by GMAS and PPK co-expression system. [ABSTRACT FROM AUTHOR]

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. Key Points: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP. [ABSTRACT FROM AUTHOR]

Enzymes used for adenosine triphosphate (ATP) synthesis play important roles in energy-dependent cascade reactions in vitro. In this study, two novel polyphosphate kinase 2 (PPK2) enzymes, HbPPK2 from Hydrogenophilaceae bacterium and NdPPK2 from Nocardioides dokdonensis, were characterized for ATP synthesis with the substrate polyphosphate (polyP). The optimum temperature and pH of both purified HbPPK2 and NdPPK2 were 30 °C and 6.5. HbPPK2 and NdPPK2 retained 30% and 14% of the initial activity at 30 °C for 12 h, respectively, whereas the presence of polyP significantly enhanced the stability of enzymes. The two PPK2s preferentially catalyzed the long-chain polyP hexametaphosphate as the phosphate donor. Adenosine monophosphate could not be used by HbPPK2 and NdPPK2 to synthesize ATP, indicating that they belonged to the class I subfamily of PPK2. HbPPK2 was used for ATP regeneration to produce glutathione by a two-enzyme cascade in vitro. 47.1 ± 0.4 mM glutathione was synthesized with a productivity of 13.5 ± 0.1 mM/h. ATP was regenerated approximately 471 times in the system within 3.5 h. HbPPK2 showed potential application for ATP regeneration in cascade reaction. [ABSTRACT FROM AUTHOR]

Summary: Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Polyphosphates represent important stores of phosphate and energy, and are abundant in many pro‐ and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP‐specific dye and a polyP‐binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabidopsis cells by heterologous expression of Escherichia coli polyphosphate kinase 1 (PPK1). Over‐expression of PPK1 but not of a catalytically impaired version of the enzyme leads to severe growth phenotypes, suggesting that ATP‐dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1‐expressing Arabidopsis lines with plants expressing the polyP‐binding domain of E. coli exopolyphosphatase (PPX1c), which co‐localized with PPK1‐generated polyP granules. These granules were stained by the polyP‐specific dye JC‐D7 and appeared as electron‐dense structures in transmission electron microscopy sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono‐ and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP‐rich acidocalcisomes in the green alga Chlamydomonas reinhardtii. Significance Statement: A chemical dye and an inorganic polyphosphate‐binding domain are shown to specifically label inorganic polyphosphate granules in transgenic Arabidopsis lines and Chlamydomonas acidocalcisomes. Using these tools we show that, in contrast to many prokaryotic and eukaryotic organisms, higher plants do not seem to contain large stores of inorganic polyphosphate. [ABSTRACT FROM AUTHOR]

Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have been described likewise for Agrobacterium tumefaciens (Seufferheld, M., Vieira, M., Ruiz, F. A., Rodrigues, C. O., Moreno, S., & Docampo, R. 2003. J. Biol. Chem. 278:29971-29978). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this -proteobacterium. Unexpectedly, fluorescence microscopical investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+45 -translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located closely together and both near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1, Δppk2) were unable to form polyphosphate, but still showed cell pole-located eYFP-HppA-foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. [ABSTRACT FROM AUTHOR]

Colibactin is a genotoxin produced primarily by Escherichia coli harboring the genomic pks island (pks+ E. coli). Pks+ E. coli cause host cell DNA damage, leading to chromosomal instability and gene mutations. The signature of colibactin-induced mutations has been described and found in human colorectal cancer (CRC) genomes. An inflamed intestinal environment drives the expansion of pks+ E. coli and promotes tumorigenesis. Mesalamine (i.e., 5-aminosalycilic acid), an effective anti-inflammatory drug, is an inhibitor of the bacterial polyphosphate kinase (PPK). This drug not only inhibits the production of intestinal inflammatory mediators and the proliferation of CRC cells, but also limits the abundance of E. coli in the gut microbiota and diminishes the production of colibactin. Here, we describe the link between intestinal inflammation and colorectal cancer induced by pks+ E. coli. We discuss the potential mechanisms of the pleiotropic role of mesalamine in treating both inflammatory bowel diseases and reducing the risk of CRC due to pks+ E. coli.

Phosphate metabolism was studied to determine whether polyphosphate (polyP) pools play a role in the enhanced resistance against Cd2+ and metal-removal capacity of Cd2+-preadapted (CdPA) Methanosarcina acetivorans. Polyphosphate kinase (PPK), exopolyphosphatase (PPX) and phosphate transporter transcript levels and their activities increased in CdPA cells compared to control (Cnt) cells. K+ inhibited recombinant Ma-PPK and activated Ma-PPX, whereas divalent cations activated both enzymes. Metal-binding polyP and thiol-containing molecule contents, Cd2+-removal, and biofilm synthesis were significantly higher in CdPA cells >Cnt cells plus a single addition of Cd2+>Cnt cells. Also, CdPA cells showed a higher number of cadmium, sulfur, and phosphorus enriched-acidocalcisomes than control cells. Biochemical and physiological phenotype exhibited by CdPA cells returned to that of Cnt cells when cultured without Cd2+. Furthermore, no differences in the sequenced genomes upstream and downstream of the genes involved in Cd2+ resistance were found between CdPA and Cnt cells, suggesting phenotype loss rather than genome mutations induced by chronic Cd2+-exposure. Instead, a metabolic adaptation induced by Cd2+ stress was apparent. The dynamic ability of M. acetivorans to change its metabolism, depending on the environmental conditions, may be advantageous to remove cadmium in nature and biodigesters.

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum ( Sl PPK2). After directed evolution and computation-supported design, we found that Sl PPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of Sl PPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effectively avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications.IMPORTANCEATP regeneration is an important basic applied study in the field of cell-free biocatalysis. Polyphosphate kinase (PPK) is an enzyme tool widely used for energy regeneration during enzymatic reactions. However, the thermal stability of the PPKs reported to date that can efficiently regenerate ATP is usually poor, which greatly limits their application. In this study, the thermal stability of a difficult-to-engineer PPK from Sulfurovum lithotrophicum was improved, guided by an ancestral sequence reconstruction strategy. The optimal variant has a 4.5-fold longer half-life at 60°C than the wild-type enzyme, thus enabling the extension of the temperature boundary for ATP regeneration. The ability of this variant to regenerate ATP was well demonstrated during high-temperature enzymatic production of nucleotides., Competing Interests: The authors declare no conflict of interest.

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2′3′-cGAMP) formation was successfully developed. The 2′3′-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2′3′-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2′3′-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.

Phosphate metabolism was studied to determine whether polyphosphate (polyP) pools play a role in the enhanced resistance against Cd2+ and metal-removal capacity of Cd2+-preadapted (CdPA) Methanosarcina acetivorans. Polyphosphate kinase (PPK), exopolyphosphatase (PPX) and phosphate transporter transcript levels and their activities increased in CdPA cells compared to control (Cnt) cells. K+ inhibited recombinant Ma-PPK and activated Ma-PPX, whereas divalent cations activated both enzymes. Metal-binding polyP and thiol-containing molecule contents, Cd2+-removal, and biofilm synthesis were significantly higher in CdPA cells >Cnt cells plus a single addition of Cd2+>Cnt cells. Also, CdPA cells showed a higher number of cadmium, sulfur, and phosphorus enriched-acidocalcisomes than control cells. Biochemical and physiological phenotype exhibited by CdPA cells returned to that of Cnt cells when cultured without Cd2+. Furthermore, no differences in the sequenced genomes upstream and downstream of the genes involved in Cd2+ resistance were found between CdPA and Cnt cells, suggesting phenotype loss rather than genome mutations induced by chronic Cd2+-exposure. Instead, a metabolic adaptation induced by Cd2+ stress was apparent. The dynamic ability of M. acetivorans to change its metabolism, depending on the environmental conditions, may be advantageous to remove cadmium in nature and biodigesters. [ABSTRACT FROM AUTHOR]

d-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using d-psicose epimerase and l-rhamnulose kinase, a method enabling high conversion of d-allulose from d-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% d-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM d-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of d-fructose into d-allulose at reduced ATP cost. [ABSTRACT FROM AUTHOR]

Abstract Burkholderia pseudomallei is a Gram negative bacterium and the causative agent of melioidosis. Nonetheless, how virulence factors and pathogenic mechanisms are regulated have been elusive. In this study, we determined a role of polyphosphate kinase 1 (Ppk1) in regulation of quorum sensing (QS) and the sigma factor RpoS, and identified genes co-regulated by Ppk1, QS and RpoS. We find that Ppk1 positively controls autoinducer production and expression of rpoS transcript. Proteomic analysis identified 70 protein spots that are differentially expressed between B. pseudomallei wildtype and its ppk1 -deficient strain. Within Ppk1regulated proteins, expression of 31 proteins are co-regulated by both RpoS and QS, whose functions of the majority of these proteins are associated with energy production and stress response. Moreover, expression of proteins involved in type III secretion system (T3SS) is also controlled by Ppk1. Quantitative PCR analysis confirmed that the T3SS genes bipB , bsaR and hrpK are down-regulated in ppk1 mutant. In addition, the ppk1 -deficient strain exhibits defects in adhesion and invasion into human lung epithelial cells. Our work therefore reveals regulation of virulence factors and a regulatory mechanism of RpoS and QS by Ppk1, which altogether participate in gene expression control, and might be crucial for pathogenicity of B. pseudomallei. Significance Polyphosphate kinase1 (Ppk1), which is a key enzyme in polyphosphate biosynthesis, is pivotal for virulence of the melioidosis pathogen B. pseudomallei. This enzyme is not present in human. Therefore, it has been proposed to be a key target for anti-bacterial drugs. An important step toward development of novel antibiotics and therapeutic strategies is an analysis of proteins that are controlled by Ppk1. By using proteomics, we find that Ppk1 co-regulates virulence-associated genes together with quorum sensing (QS) and the sigma factor RpoS. Moreover, we reveal that Ppk1 is critical for bacterial adhesion and host cell invasion, supporting the finding from our proteome analysis. Graphical abstract Unlabelled Image Highlights • Polyphosphate kinase 1 (Ppk1) controls quorum sensing and RpoS in B. pseudomallei. • Ppk1-regulated proteins include proteins associated with stress response and virulence factors. • Ppk1 is important for bacterial adhesion and host cell invasion. [ABSTRACT FROM AUTHOR]

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides.

The synthesis of the inorganic polymer polyphosphate (poly-P) in bacteria has been linked to stress survival and to the capacity of some strains to sequester heavy metals. In addition, synthesis of poly-P by certain strains of probiotic lactobacilli has been evidenced as a probiotic mechanism due to the homeostatic properties of this compound at the intestinal epithelium. We analyzed the link between poly-P synthesis, stress response, and mercury toxicity/accumulation by comparing wild-type strains of Lactobacillus and their corresponding mutants devoid of poly-P synthesis capacity (defective in the poly-P kinase, ppk, gene). Results showed that resistance to salt (NaCl) and acidic (pH 4) stresses upon ppk mutation was affected in Lactobacillus casei, while no effect was observed in two different Lactobacillus plantarum strains. Inorganic [Hg(II)] and organic (CH3Hg) mercury toxicity was generally increased upon ppk mutation, but no influence was seen on the capacity to retain both mercurial forms by the bacteria. Notwithstanding, the culture supernatants of ppk-defective L. plantarum strains possessed a diminished capacity to induce HSP27 expression, a marker for cell protection, in cultured Caco-2 cells compared to wild-type strains. In summary, our results illustrate that the role of poly-P in stress tolerance can vary between strains and they reinforce the idea of probiotic-derived poly-P as a molecule that modulates host-signaling pathways. They also question the relevance of this polymer to the capacity to retain mercury of probiotics.