High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain

Lee, Hyeon-Cheol; Kim, Jin Sook; Jang, Wonhee; Kim, Sang Yong

doi:10.1016/j.jbiotec.2010.06.011

Cited by 72 publications

(80 citation statements)

References 30 publications

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“…One volume of C. glutamicum culture was mixed with 2 volumes of 60% methanol plus 70 mM HEPES to quench the intracellular metabolism according to the methods described in the literature (Faijes et al, 2007;Lee et al, 2010). After that, cells were harvested by centrifugation and washed with ice-cold 0.9% NaCl solution three times.…”

Section: Strainsmentioning

confidence: 99%

Enhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB

Yamauchi¹,

Hirasawa²,

Nishii³

et al. 2014

J. Gen. Appl. Microbiol.

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Some microorganisms, such as Escherichia coli, harbor transhydrogenases that catalyze the interconversion between NADPH and NADH. However, such transhydrogenase genes have not been found in the genome of a glutamic acid-producing bacterium Corynebacterium glutamicum. In this study, the E. coli transhydrogenase genes udhA and pntAB were introduced into the C. glutamicum wild-type strain ATCC 13032, and the metabolic characteristics of the recombinant strains under aerobic and microaerobic conditions were examined. No major metabolic changes were observed following the introduction of the E. coli transhydrogenase genes under aerobic conditions. Under microaerobic conditions, significant metabolic change was not observed following the introduction of the udhA gene. However, the specific production rates of lactic acid, acetic acid, and succinic acid, and the overall production levels of acetic acid and succinic acid were increased by introducing the E. coli pntAB gene. Moreover, the NADH/NAD + ratio was increased by introduction of pntAB. Our results suggest that the E. coli PntAB transhydrogenase enhances the conversion of NADPH to NADH in C. glutamicum under microaerobic conditions, and the increased NADH/NAD + ratio results in increased succinic acid production. In addition, acetic acid production might be enhanced to supply ATP to the anaplerotic reaction catalyzed by pyruvate carboxylase.Key words: acetic acid; Corynebacterium glutamicum; pntAB; redox balance; succinic acid; transhydrogenases IntroductionMany oxidation and reduction reactions are involved in cellular metabolism, most of which require pyrimidine nucleotide cofactors, such as nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) and their reduced forms NADH and NADPH, to supply oxidizing and reducing equivalents. Generally, NADH is produced via catabolism, and is oxidized in the respiratory chain under aerobic conditions or by fermentation under anaerobic conditions. Anabolic reactions to produce cell biomass components, such as fatty acid and amino acid biosynthesis, require NADPH as a reducing equivalent. Therefore, maintaining the intracellular balance of NAD(P) + and NAD(P)H is very important for most intracellular metabolic reactions to occur.Some organisms contain the enzymes catalyzing the following interconversion of NADH and NADPH, called transhydrogenase:NADPH + NAD + NADP + + NADH. To date, two types of transhydrogenases have been identified; the cytoplasmic-soluble and membrane-bound types. Membrane-bound transhydrogenases have been found in the inner mitochondrial membrane of eukaryotic cells and in the cytoplasmic membrane of many bacteria, and simultaneously catalyze proton translocation across a membrane. Full PaperEnhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB (Received April 3, 2014; Accepted April 8, 2014) Yuto Yamauchi, None of the authors of this manuscript ha...

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Section: Strainsmentioning

confidence: 99%

Enhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB

Yamauchi¹,

Hirasawa²,

Nishii³

et al. 2014

J. Gen. Appl. Microbiol.

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“…Other examples include the production of thymidine, where a mutation in pgi to divert more flux through the pentose pathway and employment of a NAD kinase and transhydrogenase UdhA, each increased yields of thymidine [60]. A lactic acid bacteria based system that could convert fructose to mannitol, in high yield also employed a cofactor regeneration system [61].…”

Section: Cofactor Considerations In Metabolic Engineeringmentioning

confidence: 99%

Cofactor engineering for advancing chemical biotechnology

Wang

San

Bennett

2013

Current Opinion in Biotechnology

135

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Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.

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“…Our prior results also showed that the NADPH/NADP ratio was elevated, along with an increased NADH/NAD ratio, by the expression of soluble transhydrogenase (udhA) (7). Indeed, while the elevated NADH/NAD ratio does not easily return to the normal ratio, the elevated NADPH/NADP ratio returns readily to the normal equilibrium ratio due to the requirement for the reduction of overproduced UDP.…”

Section: Resultsmentioning

confidence: 74%

“…The overexpression of UdhA could be utilized in both the NADPH-requiring process and the process for recycling excess NADPH. We also showed that increasing the NADPH/NADP ratio, which was achieved by changing medium components and overexpressing soluble transhydrogenase (udhA) and NAD kinase (yfjB), positively affected thymidine production (6,7).…”

mentioning

confidence: 86%

“…Thymidine production in these strains has been further improved by random mutation and selection. A recent report described the production of thymidine by rationally engineered Escherichia coli strains, in which thymidine-biosynthesizing genes were amplified and thymidine-degrading genes were disrupted (5)(6)(7)(8). Studies of both constructed E. coli strains and Brevibacterium (and Corynebacterium) spp.…”

mentioning

confidence: 99%

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Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

Kim¹,

Jeong²,

Koo³

et al. 2015

Appl Environ Microbiol

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A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fedbatch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria. Thymidine is a commercially useful precursor in the chemical synthesis of various antiviral drugs, including stavudine and zidovudine (azidothymidine), the active ingredient in a formulation for the treatment of AIDS (1, 2). Thymidine, which is composed of 2-deoxyribose and a thymine base, has been produced by employing bacteria belonging to the genera Brevibacterium and Corynebacterium (3, 4). These strains were improved by chemical mutagenesis. Thymidine production in these strains has been further improved by random mutation and selection. A recent report described the production of thymidine by rationally engineered Escherichia coli strains, in which thymidine-biosynthesizing genes were amplified and thymidine-degrading genes were disrupted (5-8). Studies of both constructed E. coli strains and Brevibacterium (and Corynebacterium) spp. have used the same strategies, even though the productivities in those microorganisms were much lower than that in the E. coli recombinant strain (Ͻ500 mg/liter). The first shared strategy was the inactivation of the salvage pathway for preventing the degradation of thymidine. The second strategy was the enhancement of the reduction of nucleoside diphosphate (NDP) to deoxynucleoside diphosphate (dNDP). The third strategy was the enhancement of thymidylate synthase. In E. coli, these strategies could be achieved by deleting deoABCD, tdk, and udp with a rationally designed gene knockout and overexpression of phage T4 NDP reductase subunits and T4 thymidylate synthase (td). To reduce feedback inhibition, we overexpressed T4 thymidylate synthase and T4 NDP reductase subunits, instead of intrinsic enzymes of E. coli. In Brevibacterium (and Corynebacterium) spp., these strategies could be achieved by screening thymidine auxotrophs after chemical mutagenesis, hydroxyurea-resistant mutants, and fluorouraciland trimethoprim-resistant mutants (3, 4).Of the enzymes required for thymidine biosynthesis, NDP reductase and d...

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High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain

Cited by 72 publications

References 30 publications

Enhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB

Enhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB

Cofactor engineering for advancing chemical biotechnology

Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

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