Recent advances in poly phosphate kinase (PPK) and the construction of PPK-mediated ATP regeneration system / 生物工程学报

Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.

Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis / 生物工程学报

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

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.

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.