Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.     

Thymidine production by overexpressing NAD<Superscript>+</Superscript> kinase in an <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant strain

Intracellular NADPH/NADP⁺ ratio in cells grown on various production media with different carbon and nitrogen sources had a positive correlation with the thymidine production. To improve thymidine production in a previously engineered E. coli strain, NAD⁺ kinase was overexpressed in it resulting in the NADPH/NADP⁺ ratio shifting from 0.184 to 0.267. The [NADH + NADP⁺]/[NAD⁺ + NADPH] ratio was, however, not significantly altered. In jar fermentation, 740 mg thymidine l⁻¹ was produced in parental strain, while 940 mg l⁻¹ of thymidine was produced in NAD⁺ kinase-expressing strain.     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Biochemical properties and physiological roles of NADP-dependent malic enzyme in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate using NAD(P)⁺ as a cofactor. NADP-dependent malic enzyme (MaeB) from Escherichia coli MG1655 was expressed and purified as a fusion protein. The molecular weight of MaeB was about 83 kDa, as determined by SDS-PAGE. The recombinant MaeB showed a maximum activity at pH 7.8 and 46°C. MaeB activity was dependent on the presence of Mn²⁺ but was strongly inhibited by Zn²⁺. In order to understand the physiological roles, recombinant E. coli strains (icd ^NADP/ΔmaeB and icd ^NAD/ΔmaeB) containing NADP-dependent isocitrate dehydrogenase (IDH), or engineered NAD-dependent IDH with the deletion of the maeB gene, were constructed using homologous recombination. During growth on acetate, icd ^NAD/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icd ^NADP). Furthermore, icd ^NADP/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icd ^NAD/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icd ^NADP/ΔmaeB due to its NADP-dependent IDH. These results indicated that MaeB was important for NADPH production for bacterial growth on acetate. We also observed that MaeB activity was significantly enhanced (7.83-fold) in icd ^NAD, which was about 3-fold higher than that in icd ^NADP, when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icd ^NAD. Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.     

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD⁺ to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K _d of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD⁺ (e.g., K _d of 87 μM for FurX-NAD⁺). The kinetic data suggest that the four enzymes are efficient “furfural reductases” with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°′) for ethanol-dependent reduction of furfural was determined to be −1.1 kJ mol⁻¹. The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.     

Artificial redox coenzymes: biomimetic analogues of NAD+

A range of biomimetic analogues of the nicotinamide nucleotide coenzymes NAD(P)(H) have been developed based on the structure of a triazine dye template. These biomimetic redox coenzymes are relatively straightforward and inexpensive to synthesise and display NAD⁺-like activity with different dehydrogenases, despite their apparently minimal structural similarity to the native coenzyme NAD⁺. Horse liver alcohol dehydrogenase oxidises butan-1-ol, using the most active biomimetic coenzyme (Nap 1), with a k _cat value an order of magnitude lower and a K _m for the coenzyme two orders of magnitude higher than those using native NAD⁺. The enzymatically reduced biomimetic coenzymes may be reoxidised by phenazine methosulfate. We believe that these coenzymes may find applications in biotransformations and biosensors, and in the development of biomimetic catalysts where the redox enzyme itself is replaced by a synthetic binding site. Received: 26 October 1998 / Received revision: 25 January 1999 / Accepted: 31 January 1999     

Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum

A recently developed method for analyzing metabolic networks using ¹³C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71. Thus, in all three experiments, the flux through the PP pathway was almost constant with an average value of 69 ± 3, and the method therefore allows for a very reproducible estimation of the PP pathway flux. Phenoxyacetic acid was found to be a source of cytosolic acetyl-CoA and thereby a source of precursors for the biosynthesis of 2-aminoadipic acid, which is a central amino acid in penicillin biosynthesis. However, the labeling patterns also indicated the presence of an unrecognized pathway to cytosolic acetyl-CoA. Received: 20 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Growth rate of a non-fermentative <Emphasis Type="Italic">Escherichia coli</Emphasis> strain is influenced by NAD<Superscript>+</Superscript> regeneration

By complementing a non-fermentative Escherichia coli (ldhA ⁻ pflB ⁻) strain with the recombinant Zymomonas mobilis ethanol pathway (pdc, adhB), we evaluated the effect of different levels of enzymatic activity on growth rate demonstrating that there is a direct relationship between anaerobic growth rate and the total specific activity of pyruvate decarboxylase, which is the limiting enzyme of this specific fermentative NAD⁺ regenerating pathway. This relationship was proved to be useful to establish a selection strategy based on growth rate for the analysis of lctE libraries, which encode lactate dehydrogenase from Bacillus subtilis.     

Novel mitochondrial alcohol metabolizing enzymes of <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Ethanol is one of the most efficient carbon sources for Euglena gracilis. Thus, an in-depth investigation of the distribution of ethanol metabolizing enzymes in this organism was conducted. Cellular fractionation indicated localization of the ethanol metabolizing enzymes in both cytosol and mitochondria. Isolated mitochondria were able to generate a transmembrane electrical gradient (Δψ) after the addition of ethanol. However, upon the addition of acetaldehyde no Δψ was formed. Furthermore, acetaldehyde collapsed Δψ generated by ethanol or malate but not by D-lactate. Pyrazole, a specific inhibitor of alcohol dehydrogenase (ADH), abolished the effect of acetaldehyde on Δψ, suggesting that the mitochondrial ADH, by actively consuming NADH to reduce acetaldehyde to ethanol, was able to collapse Δψ. When mitochondria were fractionated, 27% and 60% of ADH and aldehyde dehydrogenase (ALDH) activities were found in the inner membrane fraction. ADH activity showed two kinetic components, suggesting the presence of two isozymes in the membrane fraction, while ALDH kinetics was monotonic. The ADH Km values were 0.64–6.5 mM for ethanol, and 0.16–0.88 mM for NAD⁺, while the ALDH Km values were 1.7–5.3 μM for acetaldehyde and 33–47 μM for NAD⁺. These novel enzymes were also able to use aliphatic substrates of different chain length and could be involved in the metabolism of fatty alcohol and aldehydes released from wax esters stored by this microorganism.     

Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP⁺, which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP⁺ and NAD⁺. One of the XDH variants utilized NADP⁺ about 4 times more efficiently than NAD⁺. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

Concerning the mechanism of increased thermogenesis in rats treated with dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) treatment of rats decreases gain of body weight without affecting food intake; simultaneously, the activities of liver malic enzyme and cytosolic glycerol-3-P dehydrogenase are increased. In the present study experiments were conducted to test the possibility that DHEA enhances thermogenesis and decreases metabolic efficiency via trans-hydrogenation of cytosolic NADPH into mitochondrial FADH₂ with a consequent loss of energy as heat. The following results provide evidence which supports the proposed hypothesis: (a) the activities of cytosolic enzymes involved in NADPH production (malic enzyme, cytosolic isocitrate dehydrogenase, and aconitase) are increased after DHEA treatment; (b) cytosolic glycerol-3-P dehydrogenase may use both NAD⁺ and NADP⁺ as coenzymes; (c) activities of both cytosolic and mitochondrial forms of glycerol-3-P dehydrogenase are increased by DHEA treatment; (d) cytosol obtained from DHEA-treated rats synthesizes more glycerol-3-P during incubation with fructose-1,6-P₂ (used as source of dihydroxyacetone phosphate) and NADP⁺; the addition of citratein vitro further increases this difference; (e) mitochondria prepared from DHEA-treated rats more rapidly consume glycerol-3-P added exogenously or formed endogenously in the cytosol in the presence of fructose-1,6-P₂ and NADP⁺.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Alterations in architecture and metabolism induced by ultraviolet radiation-B in the carragenophyte <Emphasis Type="Italic">Chondracanthus teedei</Emphasis> (Rhodophyta,Gigartinales)

The in vivo effect of ultraviolet radiation-B (UVBR) in apical segments of Chondracanthus teedei was examined. Over a period of 7 days, the segments were cultivated and exposed to photosynthetically active radiation (PAR) at 80 μmol photons m⁻² s⁻¹ and PAR + UVBR at 1.6 W m⁻² for 3 h per day. The samples were processed for electron microscopy and histochemistry; also was analyzed growth rates, mitochondrial activity, protein levels, content of photosynthetic pigments and photosynthetic performance. UVBR elicited increased cell wall thickness and accumulation of plastoglobuli, changes in mitochondrial organization and destruction of chloroplast internal organization. Compared to controls, algae exposed to PAR + UVBR showed a growth rate reduction of 55%. The content of photosynthetic pigments, including chlorophyll a and phycobiliproteins, decreased after exposure to PAR + UVBR. This result agrees with the decreased photosynthetic performance observed after exposing algae to PAR + UVBR. Irradiation also elicited increased activity of the antioxidant enzyme glutathione peroxidase and decreased mitochondrial NADH dehydrogenase activity, which correlated with the decreased protein content in plants exposed to PAR + UVBR. Taken together, these findings strongly indicate that UVBR negatively affects the architecture and metabolism of the carragenophyte C. teedei.     

NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD⁺ and NADH) and NADP(H) (i.e. NADP⁺ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD⁺-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD⁺ in macrophages. Inhibition of NAD⁺ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD⁺ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD⁺ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD⁺ levels by NAD⁺, NMN, or NADP⁺ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD⁺’s cytosolic role in the regulation of morphofunctional characteristics of macrophages.     

Gene cloning,expression, and characterization of a novel acetaldehyde dehydrogenase from <Emphasis Type="Italic">Issatchenkia terricola</Emphasis> strain XJ-2

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD⁺ as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K⁺, NH4⁺, dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and phenylmethyl sulfonylfluoride. In the presence of NAD⁺, ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k _cat value of 27.71/s and a k _cat/K _m value of 26.80 × 10³/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.     

Two δ1-pyrroline-5-carboxylate dehydrogenase isoforms are expressed in cultured Nicotiana plumbaginifolia cells and are differentially modulated during the culture growth cycle

δ¹-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography. The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl⁻ at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K _m values for the two substrates, P5C and NAD⁺, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected, while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth. Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed. Received: 23 May 1996 / Accepted: 18 December 1996     

The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (ble ^R) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum. Received: 2 November 1998 / Received revision: 15 January 1999 / Accepted: 5 March 1999