Knockout of the Erythromycin Biosynthetic Cluster Gene, eryBI, Blocks Isoflavone Glucoside Bioconversion during Erythromycin Fermentations in Aeromicrobium erythreum but Not in Saccharopolyspora erythraea

Isoflavone glucosides are valuable nutraceutical compounds and are present in commercial fermentations, such as the erythromycin fermentation, as constituents of the soy flour in the growth medium. The purpose of this study was to develop a method for recovery of the isoflavone glucosides as value-added coproducts at the end of either Saccharopolyspora erythraea or Aeromicrobium erythreum fermentation. Because the first step in isoflavone metabolism was known to be the conversion of isoflavone glucosides to aglycones by a β-glucosidase, we chose to knock out the only β-glucosidase gene known at the start of the study, eryBI, to see what effect this had on metabolism of isoflavone glucosides in each organism. In the unicellular erythromycin producer A. erythreum, knockout of eryBI was sufficient to block the conversion of isoflavone glucosides to aglycones. In S. erythraea, knockout of eryBI had no effect on this reaction, suggesting that other β-glucosidases are present. Erythromycin production was not significantly affected in either strain as a result of the eryBI knockout. This study showed that isoflavone metabolism could be blocked in A. erythreum by eryBI knockout but that eryBI knockout was not sufficient to block isoflavone metabolism in S. erythraea.     

Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding,salicylic acid treatment,and salinity stress in safflower,Carthamus tinctorius

Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Isolation of NADPH:isoflavone oxidoreductase,a new enzyme of Pterocarpan phytoalexin biosynthesis in cell suspension cultures of Cicer arietinum

Chickpea Cicer arietinum L.) cell suspension cultures transferred into a medium containing yeast extract accumulate the phytoalexins medicarpin and maackiain. Concomitant with accumulation of the pterocarpans a new enzyme activity is induced which was characterized as NADPH:isoflavone oxidoreductase. Maximum enzyme activity was reached 16 h after transfer of cells and then activity rapidly declined. The soluble enzyme was partially purified and shown to catalyze the reduction of the isoflavone 2'-hydroxyformononetin to the isoflavanone vestitone which is an intermediate in medicarpin biosynthesis. The enzyme data suggest that 2'-hydroxylation is a prerequisite for the conversion of isoflavones to pterocarpans.     

Induction of Phenylalanine Ammonia-Lyase Activation and Isoflavone Glucoside Accumulation in Suspension-Cultured Cells of Red Bean, Vigna angularis, by Phytoalexin Elicitors, Vanadate, and Elevation of Medium pH

Treatment of suspension-cultured cells of red bean, Vigna angularis,with nigeran resulted in an accumulation of isoflavone glucosides,such as daidzein 7-O-ß-D-glucoside, daidzein 7,4'-di-O-ß-D-glucoside,and 2'-hydroxydaidzein 7,4'-di-O-ß-D-glucoside, whichwas accompanied by a transient increase in the activity of phenylalanineanimonia-lyase (PAL). Similar effects were also seen with otherphytoalexin elicitors, such as RNase A and cell wall componentsof Phytophthora megasperma var. sojae. Interestingly, the accumulation of isoflavone glucosides andthe transient increase in PAL activity were induced also byvanadate, a specific inhibitor of plasma membrane adenosinetriphosphatase. K₃PO₄ showed similar effects, but this was ascribedto the elevation of medium pH caused by adding this basic salt.In fact, merely raising the pH of the medium was found to besufficient for the induction of PAL activity. Experiments usinginhibitors showed that the induction depends on RNA and proteinsyntheses. The results are discussed in relation to the possiblemechanism of action of phytoalexin elicitors. ¹ Present address: Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University, Furocho, Chikusa, Nagoya 464, Japan.     

Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations

The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein, and daidzein together make up 0.1–0.2 % of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by X-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of Saccharopolyspora erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations, the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance also found isoflavone coproduct potential.     

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonio lyase Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged (G.W., We 630/9-7; We 630/10-1). Thanks are given to Dr. St. Kellam (Department of Plant Microbiological Sciences, University of Canterbury, New Zealand) for correcting the English.     

Profiling isoflavone conjugates in different organs of Lupinus exaltatus Zucc

The profiles of isoflavone conjugates in extracts obtained from different parts of Lupinus exaltatus Zucc. grown in Mexico were compared using HPLC-UV and HPLC-ESI/MSn. Collision-induced dissociation-MSn experiments were performed using an ion trap analyser during HPLC-ESI/MS analyses. Nineteen isoflavone conjugates were identified in samples obtained from air-dried roots, leaves, stems and inflorescences of lupin plants. It was possible to determine the structures of the studied compounds on the basis of the MS recorded. The compounds identified were di- and mono-glucosides of genistein and 2'-hydroxygenistein with a different pattern of C- and O-glycosylation. Some glucosides were acylated with malonic acid. It was not possible to establish the glycosylation sites on the basis of MS alone; however, it was possible to differentiate isoflavone C- and O-glucosides. The highest levels of isoflavones and their conjugates were detected in roots and the lowest in stems. Free aglycones were identified in roots and inflorescences but they were not found in stems and leaves.     

大豆异黄酮合成途径相关基因差异表达分析

大豆异黄酮是一种应用广泛、具有医用和保健功能的活性物质。为揭示异黄酮合成途径相关基因表达差异,本研究采用实时定量PCR技术分析相关基因在不同大豆品种、发育时期及组织部位的表达。结果发现,苯丙氨酸解氨酶基因PAL、肉桂酸羟化酶基因C4H、香豆酸辅酶A连接酶基因4CL在高异黄酮品种中豆27 R2期叶片中的表达量显著高于低异黄酮品种楚秀;查尔酮合成酶基因CHS、异黄酮合成酶基因IFS在中豆27 R8期子粒中的表达量显著高于楚秀;细胞色素还原酶基因CPR在中豆27 R7期叶片与子粒的表达量与楚秀相比显著降低。这些差异表达的基因可能是形成大豆品种异黄酮含量高低的重要原因。     

Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in Phloem Fluids of Cucumber during Systemic Acquired Resistance Is Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase Activity in Petioles and Stems

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.     

Hydrolysis of soybean isoflavone glucosides by lactic acid bacteria

Lactobacillus delbrueckii subsp. delbrueckii KCTC 1047, grown in de Man, Rogosa and Sharpe (MRS) or soymilk media, completely hydrolyzed the isoflavone glucosides, genistin and daidzin at 50 g ml^–1, into their respective aglycones, genistein and daidzein within 30 min. Other lactic acid bacteria did not produce -glucosidase, the enzyme responsible for the hydrolysis of isoflavone glucosides, when cultured in MRS medium. Glucoside-hydrolyzing activity was induced in some lactic acid bacteria when cultured in soymilk medium. These strains hydrolyzed 70–80% of genistin into genistein and 25–40% of daidzin into daidzein.     

Cross-talk interactions of sucrose and Fusarium oxysporum in the phenylpropanoid pathway and the accumulation and localization of flavonoids in embryo axes of yellow lupine

This study investigated the effects of cross-talk interactions of sucrose and infection caused by a pathogenic fungus Fusarium oxysporum f.sp. lupini on the regulation of the phenylpropanoid pathway, i.e. the level of expression of genes encoding enzymes participating in flavonoid biosynthesis, as well as cell location and accumulation of these compounds in embryo axes of Lupinus luteus L. cv. Polo. Embryo axes, both non-inoculated and inoculated, were cultured for 96 h on Heller medium with 60 mM sucrose (+Sn and +Si) or without it (−Sn and −Si). Real-time RT-PCR to assess expression levels of the flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS) were used. Sucrose alone strongly stimulated the expression of these genes. There was a very high expression level of these genes in +Si embryo axes in the early phase of infection. Signal amplification by sucrose and the infection was most intense in the 48-h +Si axes, resulting in the highest level of expression of flavonoid biosynthetic genes. In −Si tissues, the expression level of these genes increased at 48 and 72 h after inoculation relative to 24 h; however, the relative level of expression was much lower than in +Si axes, except at 72 h for PAL and CHS.Moreover, at 48 h of culture, considerably higher activity of CHI (EC 5.5.1.6) was observed in axes with a high level of sucrose than in those with a sucrose deficit. CHI activity in +Si axes at 48 and 96 h post-inoculation was over 1.5 and 2 times higher than that in +Sn axes, as well as higher than in −Si axes.Observations of yellow lupine embryo axes under a confocal microscope showed an increased post-infection accumulation of flavonoids, particularly in cells of embryo axes infected with F. oxysporum and cultured on a medium containing sucrose (+Si). Up to 48 h post-infection in +Si axes, a very intensive emission of green fluorescence was observed, indicating high accumulation of these compounds in whole cells. Moreover, a nuclear location of flavonoids was recorded in cells. Strong staining of flavonoid end products in +Si embryo axes was consistent with the expression of PAL, CHS, CHI and IFS.These results indicate that, in the early phase of infection, the flavonoid biosynthesis pathway is considerably enhanced in yellow lupine embryo axes as a strong signal amplification effect of sucrose and the pathogenic fungus F. oxysporum.     

Sucrose-induced lupine defense against Fusarium oxysporum. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum.

Defense responses to inoculation with Fusarium oxysporum SCHLECHT f. sp. lupini were studied in embryo axes of Lupinus luteus L. cv. Polo cultured on a medium with sucrose (60 mM) or without it. Exogenous sucrose caused a marked endogenous increase in concentrations of sucrose, glucose and fructose in embryo axes. In axes cultured with sucrose, high performance liquid chromatography (HPLC) revealed generally higher levels of isoflavone glycosides (particularly until 48 h of culture) and free aglycones (genistein, wighteone, luteone). Inoculation resulted in a considerable decline in soluble carbohydrates between 24 and 72 h of culture. Simultaneously, the infection stimulated an increase in the level of free isoflavone aglycones in inoculated embryo axes, as compared to non-inoculated ones. Concentrations of free aglycones (i.e. genistein, wighteone and luteone) after infection were particularly high in inoculated embryo axes fed with sucrose. Genistein was a better inhibitor to F. oxysporum growth than genistein 7-O-glucoside tested. Exogenous sucrose also stimulated the activity of phenylalanine ammonialyase (PAL, EC 4.3.1.5)--an important enzyme initiating phenylpropanoid metabolism. After infection of tissues, a strong increase was observed in the activity of PAL and beta-glucosidase (EC 3.2.1.21)--an enzyme hydrolyzing isoflavone glycosides. Furthermore, the growth of inoculated embryo axes cultured with sucrose was less inhibited as a result of infection than inoculated axes cultured under carbohydrate deficiency conditions. Additionally, it had been reported previously that disease symptoms of embryo axes growing in the presence of sucrose were less intensive [30]. These results suggest that soluble sugars are involved in the mechanism of resistance, as they can stimulate phenylpropanoid metabolism and contribute to the increase in concentration of isoflavonoids, which are important elements of the defense system of legumes.     

大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。