Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.     

Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions

Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH₂PO₄ (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg²⁺, Mn²⁺, Ca²⁺ and Fe³⁺ ions and inhibited by Zn²⁺ and Cd²⁺ ions while Phy II activity was moderately stimulated by Fe³⁺ ions and was inhibited by Hg²⁺, Mn²⁺ and Zn²⁺ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.     

Increasing glutaminase activity in shoyu koji using a mixed culture of two koji moulds

For improved fermentation of shoyu (soy sauce), a useful koji-making system has been developed using a mixed tane-koji of two shoyu koji moulds, namely Aspergillus oryzae K2 (length of conidiophores about 350 m) and the late-conidiation strain, A. oryzae HG (length of conidiophores about 2500 m). The mixed culture of strains K2 and HG had about twice the glutaminase activity of the single-strain cultures. In addition, the number of conidia in the mixed culture was about 10% of that in a culture of strain K2 alone.     

Phytase isozymes from Aspergillus niger NCIM 563 under solid state fermentation: Biochemical characterization and their correlation with submerged phytases

Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Expression of fungal phytase on the cell surface ofSaccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast,Saccharomyces cerevisiae by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) ofAspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast α-agglutinin, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced intoS. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.     

Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1

Bacterial leaf blight (BB) is a worldwide destructive rice disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo). A novel strain of Lysobacter antibioticus, which was isolated from the rhizosphere of rice in Yunnan Province of China, can significantly inhibit the growth of various phytopathogenic bacteria and fungi, especially BB pathogen Xoo. In greenhouse experiments, whole bacterial broth culture (WBC) of strain 13-1 was more effective in reducing BB than other components of the culture, with disease suppression efficiency up to 69.7%. However, bacterial cells re-suspended in water, cell-free culture extracts, and heated cultures also significantly reduced BB severity. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments and from 57.6% to 31.7% when the WBC of strain 13-1 (10⁸ CFU/mL) was applied at 3 days and 7 days prior to pathogen inoculation, respectively. In three field trials, strain 13-1 reduced BB incidence by 73.5%, 78.3%, and 59.1%, respectively. Disease suppression by strain 13-1 varied significantly among different rice cultivars, although efficacy was not directly related to the susceptibility level of the cultivars. Efficacy of biocontrol was also affected by different pathogen isolates, with some isolates of Xoo being more sensitive to 13-1 suppression than others. These results suggest that antibiotics and density of colonization on leaves may be involved for biological control of rice BB by strain 13-1. To our knowledge, this is the first report of L. antibioticus being a potential biocontrol agent for rice bacterial blight.     

Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product

TheAspergillus niger gene encoding phytase(phyA) was expressed in canola (Brassicanapus). Phytase expression is controlled by the seed-specificcruciferin (CruA) promoter. Secretion of the enzyme was aimed for byincorporating the cruciferin signal peptide in the expression construct.Transgenic canola lines were generated by Agrobacteriummediated transformation using nptII as the selectable marker. Ninety-fiveindependent transgenic events were generated. Phytase expression in the T1seedsranged from 0 to 600 U/g seed. Single-copy lines were selected(based on segregation for kanamycin resistance, phytase expression and Southernanalyses) from originally multi-copy transgenic lines. Phytase was expressed inthese sub-lines up to 103 U/g. Expression levels were monitoredthrough an additional 3–4 generations (in the greenhouse and in thefield)and the accumulation of phytase appeared to be fairly stable. In the expressionrange studied, phytase expression was gene-dosage dependent.     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.     

High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6′-phytase, which owns high specific activity. To achieve a high expression level of 6′-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor α-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml⁻¹. The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50°C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.Electronic Supplementary MaterialSupplementary material is available for this article at     

Formation and activities of xylanhydrolysing enzymes of Humicola grisea var thermoidea

A thermophilic fungus, Humicola grisea var thermoidea, produced in liquid culture two endoxylanases (1,4--d-xylan-xylanohydrolase, EC 3.2.1.8) with M _r of 95 (Xyl I) and 13 (Xyl II) kDa. PAGE of the crude culture filtrate and of each fraction obtained by gel filtration produced three and one band, respectively. Cross-reaction of the culture filtrate and each fraction with polyclonal antibodies prepared against Xyl II produced two and one precipitin bands, respectively. Hydrolysis of wheat straw and rice husk xylan was maximal using a combination of Xyl I and Xyl II. The products formed after hydrolysis, xylo-oligosaccharides and traces of xylose, indicated an endotype enzyme action and the co-operative activities of the xylanases.     

Strain improvement and up scaling of phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 under submerged fermentation conditions

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran–3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4°C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70°C, which will be useful for its spray drying.     

Inheritance of photo-control of conidial development in the fungusBipolaris oryzae

The inheritance of light dependence for conidial development inBipolaris oryzae was analyzed using single-ascospore isolates. When a photo-induced strain was crossed with another photo-induced strain, only photo-induced progeny were produced. When a photo-induced strain was crossed with a non-photo-induced (I) strain, photo-induced and nonphoto-induced (I) progeny were produced in a ratio of 1∶1. However, when a photo-induced strain was crossed with a non-photo-induced (II) strain, a non-photo-induced (I) progeny were formed in addition to parental types. It was suggested that genes for photo-control of conidiophore induction and genes for photo-control of conidiophore maturation are located on the same chromosome.     

Identification and molecular characterization of twin-arginine translocation system (Tat) in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> strain PXO99

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. This study identified and characterized the contribution of the twin-arginine translocation (Tat) pathway to motility, chemotaxis, extracellular polysaccharide (EPS) production and virulence in X. oryzae pv. oryzae strain PXO99. The tatC disruption mutant (strain TCM) of strain PXO99 were generated, and confirmed both by PCR and Southern blotting. Strain PXO99 cells were highly motile in NYGB 0.3% soft agar plate. In contrast, the tatC mutation impaired motility. Furthermore, strain TCM cells lacked detectable flagella and exhibited almost no chemotaxis toward glucose under aerobic conditions, indicating that the Tat secretion pathway contributed to flagellar biogenesis and chemotactic responses. It was also observed that strain TCM exhibited a reductive production of extracellular polysaccharide (EPS) and a significant reduction of virulence on rice plants when compared with the wild type PXO99. However, the tatC mutation in strain PXO99 did not affect growth rate and the ability to induce hypersensitive response (HR) in nonhost tobacco (Nicotiana tabacum L. cv. Samsun). Our findings indicated that the Tat system of X. oryzae pv. oryzae played an important role in the pathogen’s virulence. L. Chen, B. Hu, and G. Qian contributed equally to this research.     

Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens

A novel, plant growth-promoting bacterium Delftia tsuruhatensis, strain HR4, was isolated from the rhizoplane of rice (Oryza sativa L., cv. Yueguang) in North China. In vitro antagonistic assay showed this strain could suppress the growth of various plant pathogens effectively, especially the three main rice pathogens (Xanthomonas oryzae pv. oryzae, Rhizoctonia solani and Pyricularia oryzae Cavara). Treated with strain HR4 culture, rice blast, rice bacterial blight and rice sheath blight for cv. Yuefu and cv. Nonghu 6 were evidently controlled in the greenhouse. Strain HR4 also showed a high nitrogen-fixing activity in N-free Döbereiner culture medium. The acetylene reduction activity and ¹⁵N₂-fixing activity (N₂FA) were 13.06 C₂H₄ nmol ml⁻¹ h⁻¹ and 2.052 ¹⁵Na.e.%, respectively. The nif gene was located in the chromosome of this strain. Based on phenotypic, physiological, biochemical and phylogenetic studies, strain HR4 could be classified as a member of D. tsuruhatensis. However, comparisons of characteristics with other known species of the genus Delftia suggested that strain HR4 was a novel dizotrophic PGPB strain.     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70–ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.     

高产杀粉蝶菌素的链霉菌鉴定及其拮抗水稻白叶枯菌活性研究

【目的】为保证农业生产可持续性发展,研发和使用环境友好的生物农药受到全社会的高度重视。微生物代谢产物农药是我国目前应用最广的生物农药,也是未来发展绿色农药的一个重要方向。【方法】利用包含水稻白叶枯菌(Xanthomonas oryzae pv. oryzae, Xoo) PXO99A的NA培养基琼脂平板,从水稻根际土壤中筛选能抑制Xoo生长的链霉菌。通过高效液相色谱和质谱分析活性代谢产物的化学结构;采用剪叶法接种Xoo到水稻叶片后,再喷施杀粉蝶菌素溶液(0.1 g/L),2周后测定叶枯症状;采用响应面分析法优化高产杀粉蝶菌素的发酵培养基;采用PacBio SMRT测序平台+Illumina HiSeq X Ten平台开展全基因组测序。平均核苷酸一致性(average nucleotide identity,ANI)用于比较HSW2009与其他链霉菌在全基因组水平的亲缘关系。【结果】分离到一株对Xoo生长有强抑制活性的链霉菌HSW2009,其活性代谢产物为杀粉蝶菌素A1(piericidin A1,简称PIE);喷施PIE可以减轻Xoo在水稻叶片内的侵染;优化HSW2009高产PIE的发...