Bioreduction of 2-azido-1-arylethanones mediated by Geotrichum candidum and Rhodotorula glutinis

Enantioselective reductions of p-X-C₆H₄C(O)CH₂N₃ (X = H, Cl, Br, CH₃, OCH₃) mediated by Rhodotorula glutinis and Geotrichum candidum afforded the corresponding alcohols with complementary R and S configurations, respectively, in excellent yield and enantiomeric excesses. The obtained (R)-azidoalcohols are important starting materials for preparation of natural products and valuable pharmaceutical compounds such as (R)-Tembamide and (R)-Aegeline.     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases, the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique. The recombinant plasmid containing mutant E83V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS115. Comparison experiments of the mutant PEL-E83V-GS and the wild-type PEL-GS showed that the optimum temperature (45°C) of the mutant was 5°C higher than that of the wild type. The thermostability of the mutant was similar to that of the wild type. The enzymatic activity of the mutant was 188 U/ml at 37°C, which was 80% that of the wild type in the same conditions. Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val, and may be responsible for the improvement of the optimum temperature. Translated from Microbiology, 2005, 32(1) (in Chinese)     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

基因组改组技术快速提高扩展青霉碱性脂肪酶产量

应用基因组改组技术快速提高扩展青霉碱性脂肪酶的产量。采用经过多代诱变的碱性脂肪酶产生菌扩展青霉(Penicillium expansum)FS8486以及分离自新疆火焰山口土样的溜曲霉(Aspergillus tamarii)FS-132作为出发菌株,经过两轮基因组改组,得到数株优良子代。其中一株酶活较出发菌株FS8486提高317%。对亲本与子代菌株的形态型、RAPD(随机扩增多态性DNA)多态性和脂肪酸组成分析初步确定筛选获得的菌株为亲本的改组子代。首次将基因组改组技术成功应用于真核微生物基因组改造,短期内使目标代谢产物获得提高,这对于在真核微生物育种中进一步推广该技术具有重要意义。     

Incorporation of the sulfur of L-[S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Burdock fructooligosaccharide enhances biocontrol of Rhodotorula mucilaginosa to postharvest decay of peaches

The influence of adding burdock fructooligosaccharide (BFO) in the culture media on the efficacy of Rhodotorula mucilaginosa in controlling postharvest decay of peaches and its possible mode of action were investigated. The antagonistic activity of R. mucilaginosa to Rhizopus decay and blue mold decay of peaches was greatly enhanced through cultivation in the nutrient yeast dextrose agar (NYDA) medium amended with BFO at the concentration of 0.32%, compared with that cultivated in NYDB without BFO. R. mucilaginosa at 1 × 10⁸ cells/mL cultivation in the NYDB media did not reduce the natural decay incidence of peaches, compared with the control after 30 d at 4 °C followed by 7 d at 20 °C. However, R. mucilaginosa cultivation in the NYDB media amended with BFO at the concentration of 0.32% reduced the natural decay incidence of peaches. The population of R. mucilaginosa harvested from NYDB amended with BFO at 0.32% increased rapidly in peach wounds compared to that harvested from NYDB without BFO no matter peaches were stored at 20 °C or 4 °C. The activities of chitinase and β-1,3-glucanase of cell-free culture filtrate of R. mucilaginosa harvested from NYDB amended with BFO at 0.32% were higher than that at other concentrations and the control.     

Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici)

Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4%, respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis, and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature. The application of P. guilliermondii is more effective for preserving chilli fruits than conventional preservation with chlorinated water.     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母(Pichia pastoris)GS115,进行异源表达。实验结果表明:该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS(627u/mL)的81%,随机突变脂肪酶PEL-ep8-GS(924u/mL)的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其Tm值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶（PEL）基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母（Pichia pastoris）GS115,进行异源表达。实验结果表明：该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS（627u/mL）的81％,随机突变脂肪酶PEL-ep8-GS（924u/mL）的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其T_m值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

Biocontrol of Botrytis cinerea in apple fruit by Cryptococcus laurentii and indole-3-acetic acid

This study evaluated the effect of a yeast antagonist Cryptococcus laurentii and a plant regulator indole-3-acetic acid (IAA) on inhibition of Botrytis cinerea infection in harvested apple fruit. The results showed that the combined treatment with C. laurentii and IAA at 20 μg/ml was a more effective approach to reduce the gray mold rot in apple wounds than the C. laurentii alone. After 4 days of incubation, gray mold incidence in the combined treatment with C. laurentii and IAA was about 18%, which was a 50% reduction in incidence compared to the treatment with C. laurentii alone. Although IAA had no direct antifungal activity against B. cinerea infection when the time interval between IAA treatment and pathogen inoculation was within 2 h, application of IAA strongly reduced gray mold infection when IAA was applied 24 h prior to inoculation with B. cinerea in apple fruit wounds. Moreover, combination of IAA and C. laurentii stimulated the activities of superoxide dismutase, catalase and peroxidase with above 1.5-fold higher than that treatment with C. laurentii alone at 48 h. Therefore, combination of C. laurentii with IAA, which integrated the dual biological activity from the antagonistic yeast and plant regulator, might be developed to be a useful approach to control gray mold in harvested apple fruit.     

Identification and characterization of chitin synthase genes in the postharvest citrus fruit pathogen Penicillium digitatum

In this study, we carried out the isolation and characterization of chitin synthase genes (CHS) of the main citrus fruit postharvest pathogen Penicillium digitatum. Using distinct sets of degenerate primers designed from conserved regions of CHS genes of yeast and filamentous fungi, PCR methods, and a DNA genomic library, five putative CHS genes (PdigCHSI, PdigCHSII, PdigCHSIII, PdigCHSV, and PdigCHSVII) were identified, isolated, sequenced, and characterized. Phylogenetic analyses, sequence identity, and domain conservation support the annotation as CHS. A very high sequence identity and strong synteny were found with corresponding regions from the genome of Penicillium chrysogenum. Gene expression of P. digitatum CHS genes during mycelium axenic growth, under oxidative and osmotic stress conditions, and during infection of citrus fruits was confirmed and quantified using quantitative RT-PCR (qRT-PCR). PdigCHSIII had the highest expression among the five genes by one order of magnitude, while PdigCHSII had the lowest. However, PdigCHSII was strongly induced coincident with conidial production, suggesting a role in conidiogenesis. The expression of PdigCHSI, PdigCHSIII, PdigCHSV, and PdigCHSVII was upregulated during infection of citrus fruit. PdigCHSV and PdigCHSVII coexpressed in most of the experiments carried out, and they are separated by a 1.77 kb intergenic region and arranged in opposite directions.     

余甘子采后软腐病菌的分离鉴定及其细胞壁水解酶活性

【背景】药食同源的余甘子果实在采后贮藏过程中极易软腐变质,严重影响其品质和经济价值。【目的】明确引起余甘子果实软腐病的病原菌种类及其生长特性和产细胞壁水解酶活性,为余甘子采后软腐病的控制及延长其贮藏期奠定基础。【方法】采用组织块分离法从采后发病的余甘子果实分离病原菌,按照科赫法则确定分离菌株的致病性;采用形态学特征结合rDNA-ITS序列分析对病原菌进行鉴定,测定病原菌菌丝生长和产孢特性,检测产胞外细胞壁水解酶活性。【结果】从具有软腐症状的余甘子果实中分离得到32株真菌,其中菌株DQ23是余甘子采后软腐病的致病菌,通过形态特征结合rDNA-ITS序列将其鉴定为Penicillium choerospondiatis。其菌丝在酵母膏葡萄糖琼脂培养基(YDA)上生长最快,在马铃薯蔗糖琼脂培养基(PSA)上产孢最多。该菌能有效利用多种碳、氮源,适宜产孢的碳源为蔗糖、葡萄糖,氮源为蛋白胨、牛肉膏、酵母膏。菌丝生长的最适温度和pH范围分别为25°C和3.0-5.0,产孢的最适温度和pH范围分别为25°C和4.0-7.0。光照均利于菌丝生长和产孢。该菌具有分解果胶、纤维素的能力,无分解蛋白质、鞣质的能力。【结论】Penicillium choerospondiatis是余甘子果实软腐病的病原菌,研究结果为该病害的防控奠定了基础。     

Controls of the expression of aspA , the aspartyl protease gene from Penicillium roqueforti

The gene (aspA) encoding the extracellular aspartyl protease from Penicillium roqueforti was cloned and characterized. Northern hybridization analyses and β-casein degradation assays revealed that aspA was strongly induced by casein in the medium and efficiently repressed by ammonia. External alkaline pH overrides casein induction, resulting in aspA repression. Cis-acting motifs known to mediate nitrogen and pH regulation of fungal gene expression are present in the aspA promoter and protein-DNA binding experiments showed that mycelial proteins interact with various regions of the promoter. Due to the efficient environmental controls on aspA expression, the promoter of aspA is an attractive candidate for the development of a controllable gene expression system in P. roqueforti. Received: 20 March 1997 / Accepted: 21 June 1997     

Biocontrol of tomato wilt by Penicillium oxalicum formulations in different crop conditions

Eight formulations of Penicillium oxalicum (FOR1 to FOR8) were obtained by the addition of various ingredients, in two separate steps of the production and drying of P. oxalicum conidia. These formulations were then evaluated against tomato wilt in three glasshouse (G1 to G3) and two field (F1 and F2) experiments. All formulations were applied to seedlings in seedbeds 7 days before transplanting at a rate of 10⁷ spores g⁻¹ seedbed substrate. The conidial viability of each formulation was estimated by measuring germination just after fluid bed-drying, before seedbed application and after 1 and 2 years of storage at 4 °C under vacuum. The densities of P. oxalicum were estimated in the seedbed substrate and in the rhizosphere of three plants per treatment just before transplanting. Initial conidial viability of formulations just after fluid bed-drying was approx. 80%, except for FOR1, FOR4, and FOR7 which were 60%. The initial viability was maintained up to 40–50% for 2 years of storage at 4 °C under vacuum, except for FOR1. All formulations had 50% viability at application time. Populations of P. oxalicum in the seedbed substrate just before transplanting were >10⁶ cfu g⁻¹ soil in G3 and F2; populations in rhizosphere were also >10⁶ cfu g⁻¹ fresh root, except for FOR3, FOR5, and FOR6 in G2. A range of 22–64% of disease reduction was observed with all formulations, although these reductions were not significant (p = 0.05) for FOR1, FOR4, and FOR5 in any experiment. Contrast analysis showed significant differences between biological treatments and untreated control (p = 0.05) in all experiments, but no significant differences between biological and chemical treatments. Initial conidial viability of P. oxalicum in formulations and populations of P. oxalicum in the seedbed substrate explained 78.26% of the variability in P. oxalicum populations in tomato rhizosphere before transplanting. Disease incidence in untreated plants was negatively correlated (r = −0.54) with the percentage of disease control. The relationship between the viability of formulations, the populations of P. oxalicum in seedbed and rhizosphere, and the control of tomato wilt is discussed.     

Production of lytic enzymes and siderophores, and inhibition of germination of basidiospores of Moniliophthora (ex Crinipellis) perniciosa by phylloplane actinomycetes

Streptomyces albovinaceus, Streptomyces caviscabies, Streptomyces griseus, Streptomyces setonii, and Streptomyces virginiae selected as antagonists of Moniliophthora (ex Crinipellis) perniciosa, the causal agent of cacao Witches’ broom, were examined in vitro to detect production of chitinases, β-1,3-glucanases, and cellulases. All the species produced chitinases, but not β-1,3-glucanases or cellulases, when grown on a liquid mineral medium containing glucose, colloidal chitin, or cell walls of M. perniciosa as a carbon source. There were no quantitative differences among species in the production of chitinase, however, the germination inhibition of basidiospores of M. perniciosa was higher when they were cultivated using glucose as a carbon source, followed by colloidal chitin and cell walls. All the species also produced hydroxymate type siderophores in similar quantities, and the quantity of siderophores did not correlate with the inhibition of basidiospore germination. The germination inhibition was more pronounced when S. albovinaceus, S. griseus, and S. virginiae were cultivated on iron-deficient medium, suggesting involvement of siderophores in the antagonism by these species of actinomycetes.     

Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community

A broad spectrum of fungal antagonists was evaluated as potential biocontrol agents (BCAs) against the soil-borne pathogen Rhizoctonia solani using a new combination of in vitro and in vivo assays. The in vitro characterisation of diverse parameters including the ability to parasitise mycelium and to inhibit the germination of Rhizoctonia sclerotia at different temperatures resulted in the selection of six potential fungal antagonists. These were genotypically characterised by their BOX-PCR fingerprints, and identified as Trichoderma reesei and T. viride by partial 18S rDNA sequencing. When potato sprouts were treated with Trichoderma, all isolates significantly reduced the incidence of Rhizoctonia symptoms. Evaluated under growth chamber conditions, the selected Trichoderma isolates either partly or completely controlled the dry mass loss of lettuce caused by R. solani. Furthermore, the antagonistic Trichoderma strains were active under field conditions. To analyse the effect of Trichoderma treatment on indigenous root-associated microbial communities, we performed a DNA-dependent SSCP (Single-Strand Conformation Polymorphism) analysis of 16S rDNA/ITS sequences. In this first assessment study for Trichoderma it was shown that the pathogen and the vegetation time had much more influence on the composition of the microbiota than the BCA treatment. After evaluation of all results, three Trichoderma strains originally isolated from Rhizoctonia sclerotia were selected as promising BCAs.     

芒果、香蕉采后病害生物防治的研究进展

芒果、香蕉采后主要病害为炭疽病、蒂腐病、冠腐病、黑腐病、黑星病.生物防治是当前芒果、香蕉采后病害控制的重要研究方向.概述了生物防治芒果、香蕉采后病害的方法,包括诱抗剂、植物提取物、拮抗微生物在芒果、香蕉采后病害防治上的研究与应用.     

Characterization of ptxS , a Pseudomonas aeruginosa gene which interferes with the effect of the exotoxin A positive regulatory gene, ptxR

The complicated process of exotoxin A production by Pseudomonas aeruginosa is controlled by several genes. We have recently described a toxA positive regulatory gene, ptxR. We also proposed the presence of another gene which is adjacent to ptxR and interferes with ptxR function on exotoxin A production. In the presence of a fragment that carries the putative gene, the enhancement in exotoxin A production by ptxR was reduced threefold. In this study, we describe the characterization of this gene. Nucleotide sequence analysis of the 2.1-kbp fragment at the 5′ end of ptxR revealed the presence of an open reading frame designated ptxS (the gene next to ptxR) which encodes a 37.4-kDa protein. The gene ptxS is transcribed in the opposite orientation to ptxR from the other DNA strand. The deduced amino acid sequence of ptxS exhibited a strong homology to several proteins of the GalR-LacI family of repressors. A putative helix-turn-helix DNA binding motif was identified at the amino-terminus region of PtxS. When PtxS was overexpressed in Escherichia coli using the T7 expression system, a single protein of 38-kDa molecular weight was detected. An isogenic mutant defective in ptxS was constructed in PAO1 using the gene replacement technique. The loss of ptxS resulted in a twofold increase in exotoxin A production compared to PAO1. The effect of ptxS on ptxR was examined using a ptxR-lacZ fusion. In the presence of ptxS, the level of β-galactosidase activity produced by the ptxR-lacZ fusion was significantly reduced. These results suggest that ptxS encodes a protein which negatively regulates ptxR expression in P. aeruginosa. Received: 29 September 1997 / Accepted: 22 December 1997     

马齿苋内生菌橘青霉和波兰青霉中抗青枯菌的活性物质

为了挖掘农作物病害生物防治新资源,以药用植物马齿苋(Portulaca oleracea)为材料,通过培养基种植法分离和纯化其根、茎、叶中的内生菌,以青枯菌(Ralstonia solanacearum)的抑菌试验评价其活性,采用菌落形态观察和ITS序列分析鉴定菌种。结果表明,从马齿苋筛选出2种具有抑制青枯菌的内生菌橘青霉(Penicillium citrinum)和波兰青霉(P. polonicum),采用液相与四极杆飞行时间串联质谱(UPLC-QTOF-MS)鉴定2种内生菌的主要活性物质为橘霉素,其对青枯菌的抑制效果比链霉素更好。因此,这为植物青枯病的生物防治提供科学依据。     

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH – and temperature optima of 3.9 and 50°C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-α-1,4-polygalacturonase (exo-PG I). K_m and V_max for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U × mg⁻¹, respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.