l-glutamate inhibits blue mould caused by Penicillium expansum in apple fruits by altering the primary nitrogen and carbon metabolisms

Although several studies have shown the ability of l -glutamate (glutamate) to mitigate the stress imposed by pathogens, the underlying mechanism is still in its infancy. To gain further knowledge, this study focussed on the effect of glutamate on primary nitrogen and carbon metabolisms during apple-Penicillium expansum interaction. The obtained result showed that glutamate could effectively restrict blue mould rot development in apples, but had no direct impact on fungal growth in vitro. The application of glutamate increased the level of nitrogen in apples, resulting in a disrupted balance of carbon and nitrogen. Consistently, the key enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) involved in the GS/GOGAT cycle, and the deaminating activity of glutamate dehydrogenase (GDH), forming 2-oxoglutarate and ammonium from glutamate, were promptly stimulated by glutamate. Interestingly, glutamate led to considerable consumption of the tricarboxylic acid (TCA) cycle intermediates, such as isocitric acid and citric acid, accompanied by the enhancement of malate dehydrogenase and succinate dehydrogenase activities. Collectively, exogenous application of glutamate might confer blue mould resistance in apples, at least in part, by redirecting host’s primary nitrogen and carbon metabolisms, such as the activation of the GS/GOGAT cycle, deaminating activity of GDH and the TCA cycle.     

Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H⁺-ATPase (0·9–1·1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca²⁺- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.     

C- and N-catabolic utilization of tricarboxylic acid cycle-related amino acids by Scheffersomyces stipitis and other yeasts

Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L-amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C. maltosa, Pichia capsulata and Yarrowia lipolytica and in basidiomycetous species such as Rhodotorula rubra and Trichosporon beigelii. Glutamate was a very efficient carbon source for Sc. stipitis, which enabled a high biomass yield/mole, although the growth rate was lower when compared to growth on glucose medium. The cells secreted waste ammonium during growth on glutamate alone. In Sc. stipitis cultures grown in glucose medium containing glutamate as the nitrogen source the biomass yield was maximal, and ethanol concentration and specific ethanol formation rate were significantly higher than in glucose medium containing ammonium as the nitrogen source. Mainly C-assimilation of glutamate but also N-assimilation in glucose-containing medium correlated with enhanced activity of the NAD-dependent glutamate dehydrogenase 2 (GDH2). A Δgdh2 disruptant was unable to utilize glutamate as either a carbon or a nitrogen source; moreover, this disruptant was also unable to utilize aspartate as a carbon source. The mutation was complemented by retransformation of the GDH2 ORF into the Δgdh2 strain. The results show that Gdh2p plays a dual role in Sc. stipitis as both C- and N-catabolic enzyme, which indicates its role as an interface between the carbon and nitrogen metabolism of this yeast.     

Screening various Yarrowia lipolytica strains for citric acid production

Citric acid (CA) productivity by Yarrowia lipolytica dependents on strain type, carbon source, carbon to nitrogen (C/N) molar ratio as well as physicochemical conditions (pH, temperature, oxygen transfer rate, etc.). In the current study, 10 different Y. lipolytica strains were first challenged in shake-flask culture for CA production in a glucose-based media under nitrogen-limited conditions. For the most promising one, strain K57, CA productivity was monitored during culture in batch bioreactor for three initial C/N molar ratio (167, 367, and 567 Cmol/Nmol). The highest CA yield (0.77 g/g glucose), titre (72.3 g/L CA), and productivity (0.04 g/g.h) were found for C/N ratio of 367. However, the highest growth rate was obtained for an initial C/N ratio of 167. From these results, Y. lipolytica strain K57 could be considered to produce CA at higher titre on glucose-based medium in batch bioreactor than others Y. lipolytica strain reported in the literature.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.     

Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane-utilizing yeast Yarrowia lipolytica

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.     

Optimization of cyclopropane fatty acids production in Yarrowia lipolytica

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long-term resistance to oxidization and low-temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large-scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed-batch fermentation set-up enable us to reach a specific productivity of 0.032 g·L⁻¹·hr⁻¹ with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

不同小白菜品种硝酸盐含量、氮代谢关键酶活性及NRT1表达和亚细胞定位

采用液体培养实验研究了不同硝铵比（c（NO3 － ）∶c（NH4 ＋）分别为50∶50、75∶25和100∶0）对低硝酸盐富 集品种‘香港特选奶白菜’和高硝酸盐富集品种‘揭农四号春白菜’硝酸盐含量、氮代谢关键酶活性的影响,并 探讨了两个品种小白菜硝酸盐转运蛋白基因的表达和亚细胞定位。结果表明：‘香港特选奶白菜’和‘揭农四号春 白菜’在硝铵比100∶0处理中,叶硝酸盐含量分别较硝铵比50∶50处理增加了13.2%和30.4%,叶柄硝酸盐含量增加 了14.3%和4.9%。随着硝铵比的增加,小白菜叶片硝酸还原酶、谷氨酸合成酶活力呈降低趋势,亚硝酸还原酶活力 呈上升趋势,谷氨酸脱氢酶活力呈先增加后降低趋势,且两个小白菜品种之间差异显著。低亲和硝酸盐转运蛋白 （nitrate transporter,NRT）1基因NRT1在两个小白菜品种中均显著表达,且高富集硝酸盐品种“揭农四号春白菜” 的表达量显著高于低富集硝酸盐品种‘香港特选奶白菜’的表达量。NRT1是一个定位于细胞膜上的低亲和NRT。     

Transglutaminase and tyrosinase as potential cross-linking tools for the improvement of rheological properties of gluten-free amadumbe dough

Gluten-free bread remains of poor quality despite efforts to amend gluten-free flours with ingredients such as hydrocolloids and proteins. Enzymatic modification of the proteins in dough may result in polymers that mimic gluten. This research investigated the effects of transglutaminase and tyrosinase on the rheological properties of amadumbe dough. Tyrosinase oxidation resulted in a 7.7–39.4% decrease in dough-free amine, and a 16.8–46.3% decrease in the dough thiol content as activity was increased from 0 to 80 U g⁻¹ flour. Transglutaminase treatment decreased the dough-free amino groups by 10–38.1% as activity was increased from 0 to 2 U g⁻¹ flour. Evidence of tyrosinase and transglutaminase-mediated cross-linking was provided by relevant model reactions monitored by mass spectrometry. An increase in dough G′ and G″ showed that both transglutaminase and tyrosinase improved dough viscoelasticity. The increase in the viscoelasticity of the dough potentially improves carbon dioxide retention during proofing.     

On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes

In the last years several reports have reported the capacity of the yeast Dekkera (Brettanomyces) bruxellensis to survive and adapt to the industrial process of alcoholic fermentation. Much of this feature seems to relate to the ability to assimilate limiting sources of nutrients, or somehow some that are inaccessible to Saccharomyces cerevisiae, in particular the sources of nitrogen. Among them, amino acids (AA) are relevant in terms of beverage musts, and could also be important for bioethanol. In view of the limited knowledge on the control of AA, the present work combines physiological and genetic studies to understand how it operates in D. bruxellensis in response to oxygen availibility. The results allowed separation of the AA in three groups of preferentiality and showed that glutamine is the preferred AA irrespective of the presence of oxygen. Glutamate and aspartate were also preferred AA in anaerobiosis, as indicated by the physiological data. Gene expression experiments showed that, apart from the conventional nitrogen catabolic repression mechanism that is operating in aerobiosis, there seems to be an oxygen‐independent mechanism acting to overexpress key genes like GAP1, GDH1, GDH2 and GLT1 to ensure adequate anaerobic growth even in the presence of non‐preferential nitrogen source. This could be of major importance for the industrial fitness of this yeast species.     

The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose

The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg²⁺-dependent dephosphorylation of phosphatidate to produce diacylglycerol, which can be acylated to form triacylglycerol (TAG). In the model oleaginous yeast Yarrowia lipolytica, TAG is the major lipid produced, and its biosynthesis requires a continuous supply of diacylglycerol, which can be provided by the PAP reaction. However, the regulation of Pah1 has not been studied in detail in Y. lipolytica, and thus its contribution to the biosynthesis of TAG in this yeast is not well understood. In this work, we examined the regulation of the PAH1-mediated PAP activity and Pah1 abundance and localization in cells growing on glucose. We found that Pah1 abundance and localization were regulated in a growth-dependent manner, yet the loss of Pah1 did not have a major effect on PAP activity. We also examined the effects of the Y. lipolytica pah1Δ mutation on cell physiology and lipid biosynthesis. The lack of Pah1 in the pah1Δ mutant resulted in a moderate decrease in TAG levels and an increase in phospholipid levels. These results showed that Pah1 contributed to TAG biosynthesis in Y. lipolytica but also suggested the presence of other activities in the pah1Δ mutant that compensate for the loss of Pah1. Also, the levels of linoleic acid were elevated in pah1Δ cells with a concomitant decrease in the oleic acid levels suggesting that the pah1Δ mutation affected the biosynthesis of fatty acids.     

Characteristics of Aspergillus niger xylanases produced on rice husk and wheat bran in submerged culture and solid‐state fermentation for an applicability proposal

Xylanolytic rich filtrates were obtained by A. niger sp in both submerged and solid‐state culture using rice husk or wheat bran as the only carbon source. Filtrates obtained on rice husk showed the highest activities (~6500 and 5200 U g^?1, respectively). Independent of carbon source, these filtrates were very stable in an acidic pH range (4–7) and mild temperatures, with high half‐life time values (more than 7 h at 50 °C) in the corresponding inactivation kinetic models. Also the effect of different metallic ions and denaturing substances was verified finding that these enzymes are not metaloproteins, and metals as Hg²⁺ and Pb²⁺ caused the greatest loss of xylanolytic activity (not higher than 30%). Xylanases produced by this A. niger strain showed important features that make them potential candidates for applications on human and livestock food industries.     

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5‐fold with the addition of Mg²⁺ (1 mm ) in the assay mixture, which means that most of the PAP activity was due to Mg²⁺‐dependent PAP enzymes (e.g. Pah1, App1). In contrast, N‐ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM‐sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth‐dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd.     

An efficient method for production of kynurenic acid by Yarrowia lipolytica

Kynurenic acid (KYNA) is a compound derived from the tryptophan catabolic pathway. Antioxidant and neuroprotective properties have been confirmed for KYNA, which makes it an interesting and important metabolite of biomedical significance. In the present study, the yeast Yarrowia lipolytica was tested for KYNA biosynthesis. The results showed that Y. lipolytica strain S12 is able to produce KYNA in high concentrations (up to 21.38 μg/ml in culture broth and 494.16 μg/g cell dry weight in biomass) in optimized conditions in a medium supplemented with tryptophan. Different conditions of culture growth, including the source of carbon, its concentration and pH value of the medium, as well as the influence of an inhibitor or precursor of KYNA synthesis, were analysed. The obtained data confirmed the presence of KYNA metabolic pathway in the investigated yeast. To our best knowledge, this is the first study that reports KYNA production in the yeast Y. lipolytica in submerged fermentation.     

Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471

Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50–55 °C, and the enzyme was stable from pH 5 to 10 and up to 37 °C. The SMC activity requires Mg²⁺ or Mn²⁺ and the order of potency to enhance the activity was Zn²⁺ ≥ Mn²⁺ > Cu²⁺ ≥ Fe²⁺. Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H₂N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.