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1.  Comparative assessment of native and heterologous 2-oxo acid decarboxylases for application in isobutanol production by Saccharomyces cerevisiae 
Decarboxylation of α-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA.
To eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a ‘decarboxylase-negative’ (pdc1Δ pdc5Δ pdc6Δ aro10Δ) S. cerevisiae background. Kinetic analyses in cell extracts revealed a superior Vmax/Km ratio of KdcA for α-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with α-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of α-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes.
This study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0374-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4665922  PMID: 26628917
Saccharomyces cerevisiae; 2-oxo acid decarboxylase; Lactococcus lactis; Isobutanol production; Fusel alcohol production
2.  Effect of Fungal Colonization of Wheat Grains with Fusarium spp. on Food Choice, Weight Gain and Mortality of Meal Beetle Larvae (Tenebrio molitor) 
PLoS ONE  2014;9(6):e100112.
Species of Fusarium have significant agro-economical and human health-related impact by infecting diverse crop plants and synthesizing diverse mycotoxins. Here, we investigated interactions of grain-feeding Tenebrio molitor larvae with four grain-colonizing Fusarium species on wheat kernels. Since numerous metabolites produced by Fusarium spp. are toxic to insects, we tested the hypothesis that the insect senses and avoids Fusarium-colonized grains. We found that only kernels colonized with F. avenaceum or Beauveria bassiana (an insect-pathogenic fungal control) were avoided by the larvae as expected. Kernels colonized with F. proliferatum, F. poae or F. culmorum attracted T. molitor larvae significantly more than control kernels. The avoidance/preference correlated with larval feeding behaviors and weight gain. Interestingly, larvae that had consumed F. proliferatum- or F. poae-colonized kernels had similar survival rates as control. Larvae fed on F. culmorum-, F. avenaceum- or B. bassiana-colonized kernels had elevated mortality rates. HPLC analyses confirmed the following mycotoxins produced by the fungal strains on the kernels: fumonisins, enniatins and beauvericin by F. proliferatum, enniatins and beauvericin by F. poae, enniatins by F. avenaceum, and deoxynivalenol and zearalenone by F. culmorum. Our results indicate that T. molitor larvae have the ability to sense potential survival threats of kernels colonized with F. avenaceum or B. bassiana, but not with F. culmorum. Volatiles potentially along with gustatory cues produced by these fungi may represent survival threat signals for the larvae resulting in their avoidance. Although F. proliferatum or F. poae produced fumonisins, enniatins and beauvericin during kernel colonization, the larvae were able to use those kernels as diet without exhibiting increased mortality. Consumption of F. avenaceum-colonized kernels, however, increased larval mortality; these kernels had higher enniatin levels than F. proliferatum or F. poae-colonized ones suggesting that T. molitor can tolerate or metabolize those toxins.
PMCID: PMC4059719  PMID: 24932485
3.  Kinetic and Chemical Mechanism of α–Isopropylmalate Synthase from Mycobacterium tuberculosis 
Biochemistry  2006;45(29):8988-8999.
Mycobacterium tuberculosis α–isopropylmalate synthase (MtIPMS) catalyzes the condensation of AcCoA with α–ketoisovalerate (α–KIV) and the subsequent hydrolysis of α–isopropylmalyl-CoA to generate the products CoA and α–isopropylmalate (α–IPM). This is the first committed step in L–leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and 1H-NMR spectroscopy. The α–keto acid specificity of the enzyme is narrow and the acyl-CoA specificity is absolute for AcCoA. In the absence of α–KIV MtIPMS does not enolize the α–protons of AcCoA, but slowly hydrolyzes acyl-CoA analogs. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random Bi Bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pKa values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of α–isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate limiting, although with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of α–isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. Based on these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.
PMCID: PMC2507874  PMID: 16846242
α-Isopropylmalate Synthase; Steady-State Kinetics; Chemical Mechanism
4.  Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli▿  
Applied and Environmental Microbiology  2009;75(19):6306-6311.
A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.
PMCID: PMC2753059  PMID: 19684168
5.  Utilization of α-Keto and α-Hydroxy Analogues of Valine by the Growing Rat 
Journal of Clinical Investigation  1974;54(2):271-277.
When 70-80-g male albino rats eat a diet furnishing daily requirement of valine for optimal growth (70 μmol/g) and all other nutrients (“complete diet”), they gain weight at an average rate of 3.0 g/100 g body wt/day. When valine is removed, they lose weight at an average 2.1 g/100 g body wt/day. The growth retardation is improved or corrected by adding valine to the diet, daily weight gain being proportional to dietary valine content over a range of 0-70 μmol/g.
Addition of α-ketoisovaleric acid instead of valine to the valine-free diet also improves or corrects the growth failure. Percent efficiency of α-ketoisovaleric acid as a substitute for valine was calculated as: 100 × (micromole valine per gram diet required to produce specified growth response)/(micromole α-ketoisovaleric acid per gram diet required to produce the same response). Efficiency of the substitution is inversely related to dietary content of the keto analogue, being 80% when diet contains 17.5 μmol/g (molar equivalent of ¼ the daily requirement of valine), and 37% when diet provides 140 μmol/g (molar equivalent of twice the daily requirement of valine).
α-Hydroxyisovaleric acid also substitutes for valine. Efficiency of the substitution at the single ration tested, 70 μmol/g diet, is 45%, similar to that for the keto analogue under the same conditions.
When [1-14C]α-ketoisovaleric acid is injected intravenously, 30-80% of the administered radioactivity is exhaled as 14CO2 within 24 h. This finding suggests that inefficiency of α-ketoisovaleric acid as a substitute for valine results in part from degradation of the keto acid to isobutyric acid by branched chain dehydrogenase-decarboxylase.
Oral administration of neomycin, polymyxin, and bacitracin reduces efficiency of α-ketoisovaleric acid as a substitute for valine by ¼-½. This effect suggests that transamination of the keto acid may be performed in part by gastrointestinal microbes.
PMCID: PMC301554  PMID: 4367888
6.  Fusarium Species from Nepalese Rice and Production of Mycotoxins and Gibberellic Acid by Selected Species 
Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.
PMCID: PMC91937  PMID: 10698766
7.  Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production▿  
Applied and Environmental Microbiology  2010;76(24):8053-8061.
2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter−1) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter−1) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.
PMCID: PMC3008247  PMID: 20935122
8.  Beauvericin Production by Fusarium Species† 
Beauvericin is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and which can induce apoptosis in mammalian cells. Beauvericin is produced by some entomo- and phytopathogenic Fusarium species (Fusarium proliferatum, F. semitectum, and F. subglutinans) and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. We tested 94 Fusarium isolates belonging to 25 taxa, 21 in 6 of the 12 sections of the Fusarium genus and 4 that have been described recently, for the ability to produce beauvericin. Beauvericin was produced by the following species (with the number of toxigenic strains compared with the number of tested strains given in parentheses): Fusarium acuminatum var. acuminatum (1 of 4), Fusarium acuminatum var. armeniacum (1 of 3), F. anthophilum (1 of 2), F. avenaceum (1 of 6), F. beomiforme (1 of 1), F. dlamini (2 of 2), F. equiseti (2 of 3), F. longipes (1 of 2), F. nygamai (2 of 2), F. oxysporum (4 of 7), F. poae (4 of 4), F. sambucinum (12 of 14), and F. subglutinans (3 of 3). These results indicate that beauvericin is produced by many species in the genus Fusarium and that it may be a contaminant of cereals other than maize.
PMCID: PMC106821  PMID: 9687479
9.  Occurrence of Fusaproliferin and Beauvericin in Fusarium-Contaminated Livestock Feed in Iowa† 
Applied and Environmental Microbiology  1998;64(10):3923-3926.
Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B1 was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.
PMCID: PMC106579  PMID: 9758820
10.  Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate 
Journal of Bacteriology  1980;142(2):513-520.
Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR92-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase Km for α-ketoisovaleric acid, 6 × 10−5 M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase Km for α-ketoisovaleric acid, 1.1 × 10−3 M; transaminase B Km for α-ketoisovaleric acid, 2 × 10−3 M).
PMCID: PMC294015  PMID: 6991477
11.  Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea. 
Journal of Bacteriology  1996;178(3):780-787.
Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.
PMCID: PMC177725  PMID: 8550513
12.  Species Diversity of and Toxin Production by Gibberella fujikuroi Species Complex Strains Isolated from Native Prairie Grasses in Kansas†  
Fusarium species from agricultural crops have been well studied with respect to toxin production and genetic diversity, while similar studies of communities from nonagricultural plants are much more limited. We examined 72 Fusarium isolates from a native North American tallgrass prairie and found that Gibberella intermedia (Fusarium proliferatum), Gibberella moniliformis (Fusarium verticillioides), and Gibberella konza (Fusarium konzum) dominated. Gibberella thapsina (Fusarium thapsinum) and Gibberella subglutinans (Fusarium subglutinans) also were recovered, as were seven isolates that could not be assigned to any previously described species on the basis of either morphological or molecular characters. In general, isolates from the prairie grasses produced the same toxins in quantities similar to those produced by isolates of the same species recovered from agricultural hosts. The G. konza isolates produce little or no fumonisins (up to 120 μg/g by one strain), and variable but generally low to moderate amounts of beauvericin (4 to 320 μg/g) and fusaproliferin (50 to 540 μg/g). Toxicity to Artemia salina larvae within most species was correlated with the concentration of either beauvericin or fusaproliferin produced. Organic isolates from some cultures of G. moniliformis were highly toxic towards A. salina even though they produced little, if any, beauvericin or fusaproliferin. Thus, additional potentially toxigenic compounds may be synthesized by G. moniliformis strains isolated from prairie grasses. The Fusarium community from these grasses appears to contain some species not found in surrounding agricultural communities, including some that probably are undescribed, and could be capable of serving as a reservoir for strains of potential agricultural importance.
PMCID: PMC383103  PMID: 15066820
13.  The Transaldolase, a Novel Allergen of Fusarium proliferatum, Demonstrates IgE Cross-Reactivity with Its Human Analogue 
PLoS ONE  2014;9(7):e103488.
Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.
PMCID: PMC4116196  PMID: 25075521
14.  d-Pantothenate Synthesis in Corynebacterium glutamicum and Use of panBC and Genes Encoding l-Valine Synthesis for d-Pantothenate Overproduction 
d-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 μmol/min mg (protein)−1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes an operon. By using panC, an assay system was developed to quantify d-pantothenate. The wild type of C. glutamicum was found to accumulate 9 μg of this vitamin per liter. A strain was constructed (i) to abolish l-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to d-pantothenate. The best resulting strain has ilvA deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (ilvBNCD) and d-pantothenate (panBC) synthesis. With this strain a d-pantothenate accumulation of up to 1 g/liter is achieved, which is a 105-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the d-pantothenate-specific part of the pathway and that the availability of β-alanine is essential for d-pantothenate formation.
PMCID: PMC91285  PMID: 10223988
15.  Analysis of an avtA::Mu d1(Ap lac) mutant: metabolic role of transaminase C. 
Journal of Bacteriology  1982;150(2):739-746.
Escherichia coli can synthesize alpha-ketoisovalerate, the precursor of valine, leucine, and pantothenate, by three routes: anabolically via dihydroxyacid dehydrase and catabolically via both the branched-chain amino acid transaminase (transaminase B) and the alanine-valine transaminase (transaminase C). An E. coli K-12 mutant devoid of transaminase C (avtA) was isolated by mutagenizing an isoleucine-requiring strain devoid of transaminase B (ilvE::Tn5) with Mu d1(Ap lac) and selecting for valine-requiring derivatives which were ampicillin resistant, Lac+, able to crossfeed an ilvD mutant, and unable to grow on alpha-ketoisovalerate in place of valine. Strains defective in one, two, or all three alpha-ketoisovalerate metabolic enzymes were constructed, and their properties were analyzed. The data indicated that avtA is the structural gene for transaminase C, that transaminase C is a single enzyme species, and that the sole pathway for pantothenate biosynthesis is from alpha-ketoisovalerate. The data further showed that isoelectric inhibits the transaminase B-catalyzed deamination of valine in vivo.
PMCID: PMC216424  PMID: 7040341
16.  Wildly Growing Asparagus (Asparagus officinalis L.) Hosts Pathogenic Fusarium Species and Accumulates Their Mycotoxins 
Microbial Ecology  2015;71:927-937.
Asparagus officinalis L. is an important crop in many European countries, likely infected by a number of Fusarium species. Most of them produce mycotoxins in plant tissues, thus affecting the physiology of the host plant. However, there is lack of information on Fusarium communities in wild asparagus, where they would definitely have considerable environmental significance. Therefore, the main scientific aim of this study was to identify the Fusarium species and quantify their typical mycotoxins present in wild asparagus plants collected at four time points of the season. Forty-four Fusarium strains of eight species—Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium oxysporum, Fusarium proliferatum, Fusarium sporotrichioides, and Fusarium tricinctum—were isolated from nine wild asparagus plants in 2013 season. It is the first report of F. sporotrichioides isolated from this particular host. Fumonisin B1 was the most abundant mycotoxin, and the highest concentrations of fumonisins B1–B3 and beauvericin were found in the spears collected in May. Moniliformin and enniatins were quantified at lower concentrations. Mycotoxins synthesized by individual strains obtained from infected asparagus tissues were assessed using in vitro cultures on sterile rice grain. Most of the F. sporotrichioides strains synthesized HT-2 toxin and F. equiseti strains were found to be effective zearalenone producers.
PMCID: PMC4823322  PMID: 26687343
Fumonisins; Fungal plant pathogens; Molecular identification; Natural Fusarium populations; Trichothecenes; Zearalenone
17.  CAM Kinase IV Regulates Lineage Commitment and Survival of Erythroid Progenitors in a Non-Cell–Autonomous Manner 
The Journal of Cell Biology  2000;151(4):811-824.
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.
PMCID: PMC2169444  PMID: 11076966
Xenopus; CaM KIV; hematopoiesis; erythropoiesis; embryogenesis
18.  The d-2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis▿ † 
Journal of Bacteriology  2008;191(3):873-881.
Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding l-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (Vmax/Km ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of d-2-hydroxyacid dehydrogenases which is unrelated to the well-described d-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD+ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.
PMCID: PMC2632062  PMID: 19047348
19.  Seroepidemiology of Human Polyomaviruses 
PLoS Pathogens  2009;5(3):e1000363.
In addition to the previously characterized viruses BK and JC, three new human polyomaviruses (Pys) have been recently identified: KIV, WUV, and Merkel Cell Py (MCV). Using an ELISA employing recombinant VP1 capsid proteins, we have determined the seroprevalence of KIV, WUV, and MCV, along with BKV and JCV, and the monkey viruses SV40 and LPV. Soluble VP1 proteins were used to assess crossreactivity between viruses. We found the seroprevalence (+/− 1%) in healthy adult blood donors (1501) was SV40 (9%), BKV (82%), JCV (39%), LPV (15%), KIV (55%), WUV (69%), MCV strain 350 (25%), and MCV strain 339 (42%). Competition assays detected no sero-crossreactivity between the VP1 proteins of LPV or MCV or between WUV and KIV. There was considerable sero-crossreactivity between SV40 and BKV, and to a lesser extent, between SV40 and JCV VP1 proteins. After correcting for crossreactivity, the SV40 seroprevalence was ∼2%. The seroprevalence in children under 21 years of age (n = 721) for all Pys was similar to that of the adult population, suggesting that primary exposure to these viruses likely occurs in childhood.
Author Summary
Polyomaviruses occupy a replicative niche in animals from birds to humans. Two human polyomaviruses, BKV and JCV, were discovered in 1971 and within the last two years, three new polyomaviruses have been found in humans: KI (KIV), WU (WUV), and Merkel Cell (MCV) polyomavirus. MCV was identified in Merkel Cell carcinomas, a rare skin cancer. To date, it has not been determined what percentage of the human population is exposed to KIV, WUV, and MCV, and when initial exposure to these viruses occurs. We determined that initial exposure to KIV, WUV, and MCV occurs in childhood, similar to that for the known human polyomaviruses BKV and JCV, and that their prevalence is high. We also found evidence that a monkey virus, Lymphotropic Polyomavirus (LPV), likely has a serologically related human counterpart. Another monkey polyomavirus, SV40, was found at ∼2% prevalence, a level that does not support its role in human disease.
PMCID: PMC2655709  PMID: 19325891
20.  Sequence Variation within the KIV-2 Copy Number Polymorphism of the Human LPA Gene in African, Asian, and European Populations 
PLoS ONE  2015;10(3):e0121582.
Amazingly little sequence variation is reported for the kringle IV 2 copy number variation (KIV 2 CNV) in the human LPA gene. Apart from whole genome sequencing projects, this region has only been analyzed in some detail in samples of European populations. We have performed a systematic resequencing study of the exonic and flanking intron regions within the KIV 2 CNV in 90 alleles from Asian, European, and four different African populations. Alleles have been separated according to their CNV length by pulsed field gel electrophoresis prior to unbiased specific PCR amplification of the target regions. These amplicons covered all KIV 2 copies of an individual allele simultaneously. In addition, cloned amplicons from genomic DNA of an African individual were sequenced. Our data suggest that sequence variation in this genomic region may be higher than previously appreciated. Detection probability of variants appeared to depend on the KIV 2 copy number of the analyzed DNA and on the proportion of copies carrying the variant. Asians had a high frequency of so-called KIV 2 type B and type C (together 70% of alleles), which differ by three or two synonymous substitutions respectively from the reference type A. This is most likely explained by the strong bottleneck suggested to have occurred when modern humans migrated to East Asia. A higher frequency of variable sites was detected in the Africans. In particular, two previously unreported splice site variants were found. One was associated with non-detectable Lp(a). The other was observed at high population frequencies (10% to 40%). Like the KIV 2 type B and C variants, this latter variant was also found in a high proportion of KIV 2 repeats in the affected alleles and in alleles differing in copy numbers. Our findings may have implications for the interpretation of SNP analyses in other repetitive loci of the human genome.
PMCID: PMC4378929  PMID: 25822457
21.  Apolipoprotein(a) Kringle-IV Type 2 Copy Number Variation Is Associated with Venous Thromboembolism 
PLoS ONE  2016;11(2):e0149427.
In addition to the established association between high lipoprotein(a) [Lp(a)] concentrations and coronary artery disease, an association between Lp(a) and venous thromboembolism (VTE) has also been described. Lp(a) is controlled by genetic variants in LPA gene, coding for apolipoprotein(a), including the kringle-IV type 2 (KIV-2) size polymorphism. Aim of the study was to investigate the role of LPA gene KIV-2 size polymorphism and single nucleotide polymorphisms (SNPs) (rs1853021, rs1800769, rs3798220, rs10455872) in modulating VTE susceptibility. Five hundred and sixteen patients with VTE without hereditary and acquired thrombophilia and 1117 healthy control subjects, comparable for age and sex, were investigated. LPA KIV-2 polymorphism, rs3798220 and rs10455872 SNPs were genotyped by TaqMan technology. Concerning rs1853021 and rs1800769 SNPs, PCR-RFLP assay was used. LPA KIV-2 repeat number was significantly lower in patients than in controls [median (interquartile range) 11(6–17) vs 15(9–25), p<0.0001]. A significantly higher prevalence of KIV-2 repeat number ≤7 was observed in patients than in controls (33.5% vs 15.5%, p<0.0001). KIV-2 repeat number was independently associated with VTE (p = 4.36 x10-9), as evidenced by the general linear model analysis adjusted for transient risk factors. No significant difference in allele frequency for all SNPs investigated was observed. Haplotype analysis showed that LPA haplotypes rather than individual SNPs influenced disease susceptibility. Receiver operating characteristic curves analysis showed that a combined risk prediction model, including KIV-2 size polymorphism and clinical variables, had a higher performance in identifying subjects at VTE risk than a clinical-only model, also separately in men and women.
PMCID: PMC4762703  PMID: 26900838
22.  Diversity of Fusarium species and mycotoxins contaminating pineapple 
Journal of Applied Genetics  2013;54(3):367-380.
Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B1–B3, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB1 content was 250 μg g−1 in pineapple skin and 20 μg ml−1 in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.
Electronic supplementary material
The online version of this article (doi:10.1007/s13353-013-0146-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3720990  PMID: 23572446
Ananas comosus; FUM genes; Mycotoxins; Phylogeny; Tropical fruit diseases
23.  The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species 
BMC Genomics  2014;15(1):660.
Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).
Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.
The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-660) contains supplementary material, which is available to authorized users.
PMCID: PMC4133081  PMID: 25102932
Metarhizium anisopliae; Genome; Pathogenicity; Motifs; Mating-type; Comparative; Genomics
24.  Enriched environment treatment reverses depression-like behavior and restores reduced hippocampal neurogenesis and protein levels of brain-derived neurotrophic factor in mice lacking its expression through promoter IV 
Translational Psychiatry  2011;1(9):e40-.
Promoter IV-driven expression of brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is implicated in the pathophysiology of major depression. We previously reported that mice lacking expression of BDNF through promoter IV (BDNF-KIV mice) exhibit a depression-like phenotype. Here, we examined whether the depression-like phenotype and decreased levels of BDNF because of promoter IV deficit could be rescued by enriched environment (EE) treatment, a potential antidepressant intervention. Three weeks of EE treatment rescued depression-like behavior of BDNF-KIV mice as assessed by the tail suspension test, open-field test and sucrose preference test. EE treatment also increased BDNF transcripts driven by multiple endogenous promoters and restored BDNF protein levels in the hippocampus (HIP) of BDNF-KIV mice. Further, we investigated adult hippocampal neurogenesis as a possible cellular mechanism underlying the depression-like behavior and its recovery in BDNF-KIV mice. We found that the number of surviving progenitors and their dendritic length in the dentate gyrus of the HIP were reduced in BDNF-KIV mice compared with the control wild-type mice. EE treatment restored the reduction in cell survival and dendritic length and increased cell proliferation in BDNF-KIV mice. In conclusion, this study demonstrated that EE rescued depression-like behavior, decreased BDNF levels and defective neurogenesis in the HIP caused by lack of promoter IV-driven BDNF expression. These results suggest that decreased BDNF levels because of one impaired promoter can be compensated by other BDNF promoters and that BDNF levels may be one of the key factors regulating depression and antidepressant effects through hippocampal neurogenesis.
PMCID: PMC3309483  PMID: 22832656
BDNF; promoter; neurogenesis; enriched environment; depression; mice
25.  Production of beauvericin by a strain of Fusarium proliferatum isolated from corn fodder for swine. 
Applied and Environmental Microbiology  1994;60(10):3894-3896.
Beauvericin, a cyclodepsipeptide, was produced by cultures of three strains of Fusarium proliferatum, M-5991, M-6992, and M-6993, grown on cracked corn. M-5991 produced approximately 1,000-mg/kg levels of fumonisins, moniliformin, and beauvericin.
PMCID: PMC201906  PMID: 7986058

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