Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production.
ABSTRACT Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 +/- 0.006 mol lysine (mol glucose)(-1), which was 12% higher than that of the empty vector control strain.
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ABSTRACT: Corynebacterium glutamicum is the workhorse for the production of amino acids, including L-isoleucine (Ile). During Ile biosynthesis, NADPH is required as a crucial cofactor. In this study, four NADPH-supplying strategies based on NAD kinase, NADH kinase, glucose-6-phosphate dehydrogenase, and NAD kinase coupling with glucose-6-phosphate dehydrogenase were compared, and their influences on Ile biosynthesis were examined. PpnK is a NAD kinase of C. glutamicum ssp. lactofermentum JHI3-156 that predominantly phosphorylates NAD(+) to produce NADP(+). Pos5 is a NADH kinase of Saccharomyces cerevisiae that predominantly phosphorylates NADH to produce NADPH. Zwf is a glucose-6-phosphate dehydrogenase of JHI3-156. The ppnK, POS5, zwf, and zwf-ppnK genes were overexpressed in the Ile-producing strain JHI3-156. The expression of all four genes increased intracellular NADPH concentration and Ile production. The increase of NADPH concentration and Ile production in a POS5-expressing strain (229 and 75.6 %, respectively) was higher than that in a ppnK-expression strain. The expression of zwf also increased NADPH supply and Ile biosynthesis, but the constitutive expression of zwf was not as effective as the inducible expression of zwf. Coexpression of zwf and ppnK genes greatly enhanced NADPH supply and thus improved Ile production by up to 85.9 %, indicating that this strategy was the most effective one. These results are helpful for improving Ile biosynthesis and other biosynthetic processes.Applied biochemistry and biotechnology 07/2013; · 1.94 Impact Factor
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ABSTRACT: BACKGROUND: L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. RESULTS: A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named DeltaAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum DeltaAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum DeltaAPE6937R42. CONCLUSIONS: The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds.BMC Biotechnology 06/2013; 13(1):47. · 2.59 Impact Factor
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ABSTRACT: Shikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu(R)), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production. The pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroGfbr, aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (DeltaaroKDeltaaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5Ptac promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol. An SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production.Microbial Cell Factories 02/2014; 13(1):21. · 4.25 Impact Factor