The functions of NAD(H) (NAD⁺ and NADH) and NADP(H) (NADP⁺ and NADPH) are undoubtedly significant and distinct. Hence, regulation of the intracellular balance of NAD(H) and NADP(H) is important. The key enzymes involved in the regulation are NAD kinase and NADP phosphatase. In 2000, we first succeeded in identifying the gene for NAD kinase, thereby facilitating worldwide studies of this enzyme from various organisms, including eubacteria, archaea, yeast, plants, and humans. Molecular biological study has revealed the physiological function of this enzyme, that is to say, the significance of NADP(H), in some model organisms. Structural research has elucidated the tertiary structure of the enzyme, the details of substrate-binding sites, and the catalytic mechanism. Research on NAD kinase also led to the discovery of archaeal NADP phosphatase. In this review, we summarize the physiological functions, applications, and structure of NAD kinase, and the way we discovered archaeal NADP phosphatase.

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The relationship between the antibacterial activity and structure of 9-O,9′-O-demethyl (+)-virgatusin (Virg 3) was examined. The conversion of hydroxy groups on the 9 and 9′ positions to amino groups increased the activity. It was found that the 3′-methoxy group was more important for higher activity than the 4′-methoxy group on the 7′-phenyl group, and that the 3,4-methylenedioxy group on the 7-phenyl group was necessary for activity.

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Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16α-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16α-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16α-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11α,16α,18-triol, which is a possible metabolite of phyllocladan-16α-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.

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A xylanase gene, xyn1, which encodes Paenibacillus sp. strain W-61 xylanase 1 (Xyn1), was cloned in Escherichia coli. xyn1 encodes 211 amino acid residues, including 28 amino acid residues of a signal peptide. The deduced amino acid sequence of the mature Xyn1 showed 95.7%, 84.0%, and 83.7% identity to family 11 xylanases of Aeromonas caviae ME-1, Paenibacillus sp., and Bacillus stearothermophilus respectively. The physico-chemical properties of recombinant Xyn1 were very similar to those of intact Xyn1, except for the molecular mass. The pattern of xylooligosaccharides generated by rXyn1 was investigated by fluorophore-assisted carbohydrate electrophoresis (FACE). The degradation rate of xylohexaose by rXyn1 increased markedly as compared with that of xylopentaose. Xylohexaose had a single preferential point of cleavage by rXyn1. On the basis of the pattern of action of xylooligosaccharides, the number of subsites was estimated to be six. The catalytic site was located between the third and the fourth subsites from non-reducing end.

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5-Methylthioribulose-1-phosphate (MTRu-1-P) dehydratase catalyzes the reaction from MTRu-1-P to 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) in the methionine salvage pathway in Bacillus subtilis. The properties of this enzyme remain to be determined. We characterized these properties using a recombinant protein. The enzyme, with a molecular mass of 90 kDa, was composed of four subunits. The K_m and V_max of the enzyme were 8.9 μM and 42.7 μmole min⁻¹ mg protein⁻¹ at 25 °C respectively. Maximum activity was observed at pH 7.5 to 8.5 and 40 °C. The activation energy of the reaction from MTRu-1-P to DK-MTP-1-P was 63.5 kJ mol⁻¹. The reaction product DK-MTP-1-P was labile, and decomposed at a rate constant of 0.048 s⁻¹ to an unknown compound that was not utilized by DK-MTP-1-P enolase, the enzyme catalyzing the next step. The function of this enzyme in the pathway is discussed.

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Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.

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A Na⁺/H⁺ antiporter gene (CvNHA1) was cloned from the salt-tolerant yeast Candida versatilis. CvNHA1 encodes an antiporter with a typical yeast plasma membrane Na⁺/H⁺ antiporter structure. Transcription of CvNHA1 in C. versatilis cells was dependent on the salinity of the culture. When CvNHA1 was expressed in salt-sensitive Saccharomyces cerevisiae cells, increased salt-tolerance was observed, indicating that Cvnha1p possesses an Na⁺/H⁺ antiporter function, because the increased salt-tolerance was dependent on the extracellular pH. It appears that Cvnha1p mediates only the transport of Na⁺. In an S. cerevisiae transformant harboring a CvNHA1-EGFP fusion plasmid in which the greater part of the C-terminal hydrophilic region of Cvnha1p was deleted by fusion with enhanced green fluorescent protein (EGFP), the Cvnha1-EGFP fusion protein was localized mainly in the plasma membrane, and the NaCl-tolerance of this transformant was greater than that of a strain harboring the entire CvNHA1 gene.

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The novel amphiphilic vitamin C derivative disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), which has a C₁₈ alkyl chain attached to the stable ascorbate derivative sodium L-ascorbic acid 2-phosphate (VCP-Na), was evaluated for reduction of cell damage induced by oxidative stress, ultraviolet A (UVA), ultraviolet B (UVB), and H₂O₂; stimulation of collagen synthesis against UVA irradiation; and inhibition of matrix metalloproteinase-1 (MMP-1) activity induced by UVA in human normal dermal fibroblasts. VCP-IS-2Na pretreatment resulted in significant protection against cell damage induced by UVB, UVA, and H₂O₂. The amount of type I collagen following UVA irradiation was increased by treatment with VCP-IS-2Na in a concentration-dependent manner. These effects of VCP-IS-2Na were superior to those of L-ascorbic acid (vitamin C, VC) and VCP-Na. On the other hand, VCP-IS-2Na suppressed 65% of the excess MMP-1 irradiated UVA, and VC and VCP-Na slightly suppressed it.

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In a screening for small-molecule compounds that alleviate the deleterious effects of external CaCl₂ on zds1Δ strain yeast, we found 2-((1-(hydroxymethyl) cyclohexyl) methyl) naphthalen-1-ol (NKH-7) to be an active compound. NKH-7 also inhibited cell growth at higher concentrations. To identify its target in growth inhibition, we isolated NKH-7-resistant mutants and selected those mutants that exhibited dominant or semi-dominant resistance specifically to NKH-7. By gene cloning, a TUB1 mutant gene encoding α-tubulin with a Ser248Pro mutation was identified. Deletion of the TUB3 gene, a minor gene encoding α-tubulin, led to supersensitivity to NKH-7. Cellular tubulin-containing arrays as visualized by green fluorescent protein (GFP)-labeled α-tubulin diminished rapidly on exposure to the inhibitor. The mutation was situated proximal to the α-β interface of α-tubulin in microtubule protofilaments, suggesting the possibility that NKH-7 affects the hydrolysis of GTP bound to β-tubulin. A functional connection perhaps exists between the tubulin inhibition and Ca²⁺-dependent cell-cycle regulation.

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α and β tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum α⁄β-tubulin (CAnm α⁄β-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant α⁄β tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of α and β tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5′-triphosphate (GTP).

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Transglutaminases (TGs) are a family of enzymes that catalyze Ca²⁺-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.

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The fish pathogen Vibrio alginolyticus contains two unique flagellar systems. The LuxS quorum sensing system is reported to regulate the expression of virulence factors in a wide variety of pathogenic bacteria. Our previous work demonstrated that inactive luxS led to decreased virulence in V. alginolyticus. In this study, LuxS-dependent regulation of motility and flagella biogenesis, the potential virulence factors in V. alginolyticus, were further investigated. A luxS-deleted mutant showed deficiency in motility and flagella formation, and an intact luxS complemented the defect. Since motility is flagella dependent, V. alginolyticus flagella biogenesis related genes, including the flagella regulator genes flaK and lafK and the sub-hierarchical flagellar genes fliS and lafA, were cloned and identified. Based on quantitative real-time reverse transcription-PCR, differential expression of these genes was confirmed in wild-type and luxS mutants. Our results indicate that LuxS plays an important role in the regulation of motility and flagella biogenesis in V. alginolyticus.

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A response regulator, NblR, of the cyanobacterium Synechococcus elongatus PCC 7942 is known to induce expression of the nblA gene, a key factor in phycobilisome degradation (bleaching) under nutrient-deprivation conditions. In this study, we observed phosphorylation-independent regulation of NblR activity. We constructed a mutant strain expressing NblR (D57A), in which a putative phospho-accepting Asp-57 was replaced with Ala residue. Under nitrogen deprivation, this strain exhibited the typical bleaching phenotype observed in wild-type cells. Moreover, in the mutant, the nblA transcript accumulated at a level similar to that of the wild type. Our results indicate that activation of NblR is independent of phosphorylation, if any, by a cognate histidine kinase. Screening of proteins interacting with NblR by yeast two-hybrid analysis revealed two candidates, MreC and NarB, suggesting a novel mechanism that activates NblR, or other functions of the response regulator.

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We have found that Ebp2p is essential for maturation of 25S rRNA and assembly of 60S pre-ribosomal subunits in Saccharomyces cerevisiae. We obtained three temperature-sensitive ebp2 mutants by PCR. Polysome analysis revealed that the synthesis of 60S ribosomal subunits was compromised in each of the ebp2 mutants at the restrictive temperature. The ebp2 alleles affected the transcriptional repression of both rRNA and ribosomal protein genes due to a secretion block. Fluorescence microscopy showed that a secretion block led to condensation of nucleolar Ebp2p, whereas that was not the case with the ebp2 mutant. These results suggest that Ebp2p is implicated in the secretory response, including changes in nucleolar architecture.

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The gene for the D-mandelate dehydrogenase (D-ManDH) of Enterococcus faecalis IAM10071 was isolated by means of an activity staining procedure and PCR and expressed in Escherichia coli cells. The recombinant enzyme exhibited high catalytic activity toward various 2-ketoacid substrates with bulky hydrophobic side chains, particularly C3-branched substrates such as benzoylformate and 2-ketoisovalerate, and strict coenzyme specificity for NADH and NAD⁺. It showed marked sequence similarity with known NADP-dependent 2-ketopantoate reductases (KPR). These results indicate that together with KPR, D-ManDH constitutes a new family of D-2-hydroxyacid dehydrogenases that act on C3-branched 2-ketoacid substrates with various specificities for coenzymes and substrates.

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Bovine mammary epithelial cells (bMECs) synthesize lactoferrin, which is secreted into milk. Our results suggest that prolactin stimulated secretion of lactoferrin in primary bMECs and their clonal cell line under serum-free conditions. Prolactin also stimulated mRNA expression of lactoferrin in the clonal cell line. This effect was reduced by AG-490, suggesting that the prolactin-stimulated mRNA expression of lactoferrin was mediated by Janus kinase (JAK)2.

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Prolonged incubation with sucrose as an acceptor and cycloinulohexaose as a donor at a high concentration of cycloinulooligosaccharide fructanotransferase afforded a series of fructooligosaccharides. Their chain-length distribution extended over 10 degrees of polymerization when the donor concentration was increased to 120 mM. Increasing the acceptor concentration proved effective in improving the yield of inulin-type oligosaccharides because hydrolysis was suppressed.

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Excess thymidine induced premature senescence in normal human fibroblasts (TIG-7), with induction of typical senescence markers. Nuclear swelling, as well as cell swelling, was clearly observed in these senescent cells. Simultaneous addition of MAP kinase inhibitors, U0126, SB203580, and SP60025, effectively suppressed induction of premature senescence and senescence markers.

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This study investigated whether intake of lactoferrin (LF) would improve or prevent anemia in female long distance runners who were training during the summer season and had a high risk of iron-deficiency anemia. Sixteen female long distance runners were divided into a group taking LF and iron (the LF group) and a group that only took iron (the control group) for 8 weeks. In the control group, the ferritin, serum iron, and red blood cell count were significantly lower than before treatment. In the LF group, the hematology data showed no significant change during the 8 weeks. The red blood cell count was significantly higher in the LF group than in the control group. The blood lactate level following a 3,000-m pace run of the control group was also significantly higher than that of the LF group. These observations suggest the possibility that intake of LF increases the absorption and utilization of iron and would be useful in the prevention of iron deficiency anemia among female long distance runners.

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Dietary plant and animal peptides have been shown to reduce serum lipids. However, the potential of food-derived peptides has yet to be fully elucidated. We investigated the physiological importance of potato peptides in rats fed on a cholesterol-free diet containing 20% potato peptides (PP), when compared with two diets containing either 20% casein (CN) or 20% soy peptides (SP). The high-density lipoprotein (HDL)-cholesterol (+13.8%) and serum triglyceride (−38%) concentrations in the PP-fed group, non-HDL-cholesterol level in the PP- (−22.5%) and SP- (−15.7%) fed groups, and serum total cholesterol concentration (−12%) in the SP-fed group, were significantly different from the control group at the end of the experiment. The fecal excretion of neutral and acidic sterols was higher in the PP- and SP-fed groups, respectively, relative to the control group. These results indicate that the observed changes in the serum cholesterol levels in rats fed on soy and potato peptide appear to have been due to different mechanisms.

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We investigated the inhibitory effect of an oral administration of a hop water extract (HWE) on the development of dermatitis by using NC/Nga atopic dermatitis model mice. The induction of allergic dermatitis was conducted by tape-stripping and topical application of a mite antigen (Dermatophagoides farinae) on to the ear once a week for 10 weeks. HWE was orally administered at a dose of 100 or 500 mg/kg. The total immunoglobulin E (IgE) concentration in serum and the ear thickness were periodically examined. Finally, the antigen-specific IgE level in the serum and the production of interleukin (IL)-4, IL-12 and interferon (IFN)-γ from splenocytes and cervical lymph node cells were measured. The oral administration of HWE significantly inhibited the increase of total IgE production and ear swelling throughout the experimental period. The production of IL-12 was significantly lower in the HWE administered group than in the control group. The results suggest that the intake of HWE may be effective in preventing and alleviating the development of atopic dermatitis-like skin disease.

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The IgE-binding proteins in beer were examined by immunoblotting analysis with sera of patients sensitive to beer. Several proteins were immunoblotted with the sera, and among these, 18-kDa proteins were identified as new IgE-binding proteins in beer. Perhaps they originated from barley as a raw material.

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The methanol extract of kiwifruit leaf suppressed the postprandial blood glucose level after an oral administration of soluble starch or sucrose in mice. The mechanism of action is proposed to be due to the α-amylase-inhibiting activity in the 90% aqueous methanol fraction and α-glucosidase-inhibiting activity in the n-buthanol fraction, based on the results of in vitro experiments.

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We examined the effect of whisky congeners, substances other than ethanol in whisky, on melanogenesis in mouse B16 melanoma cells. Treatment with whisky congeners significantly blocked melanogenesis. Our results indicate that the inhibitory effects of whisky congeners on melanogenesis is due to direct inhibition of tyrosinase activity and to suppression of tyrosinase protein levels.

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The intestinal permeability of low-molecular-weight hyaluronan (LMW-HA) was investigated by using cultured monolayers of Caco-2 cells. The amount of LMW-HA that permeated the Caco-2 monolayers was measured by a carbazole assay. The permeability of LMW-HA increased inversely with the molecular size and was dose-dependent. The transport was observed to be energy-independent, and was correlated with the tight junction (TJ) permeability. These results suggest that LMW-HA permeated the Caco-2 cell monolayers via the paracellular pathway.

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Chiisanoside is the main component of Acanthopanax sessiliflorus leaves. Simultaneous administration of chiisanoside resulted in a decrease in the plasma TG level and increase of undigested TG in the intestinal lumen after oil gavage to mice. This suggests that chiisanoside has the potential to prevent obesity as a lipase inhibitor which suppresses fat absorption in vivo.

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Biocalorimetric experiments were performed to investigate the aerobic growth of Pseudomonas aeruginosa, isolated from tannery saline wastewater. Growth factors (pH, Inoculum size, carbon source, temperature, aeration rate, and agitation rate) were optimized in shaker and calorimeter based on the growth of P. aeruginosa and heat generation rates. A limiting value of 0.2% glucose concentration was found to be optimum for the growth of P. aeruginosa in a complex growth medium, and the heat flux (q_r) profiles resulting from the metabolic activity of P. aeruginosa further confirmed this observation. The bacterial growth profile was found to correlate well with the metabolic heat generated. Heat-yield values were calculated for both glucose consumption and the growth of P. aeruginosa from the calorimetric results. Metabolic shifts in substrate uptake from glucose to peptone present in growth medium was observed by the variations in heat-flux profile. The calorimetric data presented in this study should be useful in understanding the behavior of the isolated bacterial strain in degrading complex and mixed substrates commonly observed in tannery saline waste stream, and further to extend the results for scale-up studies.

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The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD⁺ as a specific electron acceptor, and the apparent K_m values for DLA and NAD⁺ were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K_m values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 °C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 °C.

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Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was also found that Asaia has a cyanide-sensitive cytochrome bo₃-type ubiquinol oxidase as sole terminal oxidase in the respiratory chain, and that it exhibits a higher H⁺/O ratio.

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Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu²⁺ solubilized from 1% chalcopyrite (CuFeS₂) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg·l⁻¹·30 d⁻¹. The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 μl O₂ uptake·mg protein⁻¹·min⁻¹. In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 μl O₂ uptake·mg protein⁻¹·min⁻¹. Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.

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Polypropylene glycol (PPG)-assimilating Sphingobium sp. strain PW-1 was grown on 0.5% PPG 700. PPG and its metabolites were analyzed by MALDI-TOF mass spectrometry. An oxidized form of a terminal alcohol group appeared with each molecular species as a metabolite. Cell-free extract showed PPG dehydrogenase activity. In this way, the oxidative metabolic pathway for PPG was confirmed.

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We isolated and characterized two small cryptic indigenous plasmids, pYAN-1 (4,896 bp) and pYAN-2 (4,687 bp), from Sphingobium yanoikuyae, and developed a versatile system that permitted genetic manipulation of the genus Sphingomonas. Nucleotide sequencing of both plasmids revealed that they contained mobA, mobs, and repA genes, which are predicted to encode proteins associated with mobilization and replication, in common. Transformation with each plasmid harboring the antibiotic resistance gene by electroporation was fully successful, using Novosphingobium capsulatum as a host.

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The QuikChange site-directed mutagenesis methodology was applied to constructing a randomly mutagenized plasmid library simply by adding manganese to the reaction mixture. This method is superior to the normally employed Pol I-type polymerase-based error-prone PCR in that (i) it does not require a subsequent ligation reaction, and (ii) there is no accumulation of mutations at the same site. α-Complementation analysis and subsequent sequence analyses of the lacZα genes in the mutated library revealed that the mutations occurred randomly within the target gene and involved all possible base substitutions.

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To determine whether intracellular ATP levels can be affected, Escherichia coli overexpressing phosphoenolpyruvate carboxykinase (pck) or phosphoenolpyruvate carboxylase (ppc) were grown in glucose minimal medium. The Pck-overexpressing cells showed approximately twice the intracellular ATP concentration, with 22% slower growth than the Ppc-overexpressing strain. This unexpected result of higher ATP coupled with slower growth is discussed based on transcriptome analysis.

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