De novo transcriptome assembly of Aureobasidium melanogenum CGMCC18996 to analyze theβ-poly(L-malic acid)biosynthesis pathway under the CaCO_(3) addition

β-Poly(L-malic acid)(PMLA)is a water-soluble biopolymer used in food,medicine and other industries.To date,the biosynthesis pathway of PMLA has not been fully elucidated.In this study,we sequenced the transcriptom e of strain Aureobasidium melanogenum under 20 g/L CaCO_(3) addition.The resulting sequencing reads were assembled and annotated for the differentially expressed genes(DEGs)analysis and novel transcripts identification.The result indicated that with the CaCO_(3) addition,the tricarboxylic cycle(TCA)cycle and glyoxylate pathway were up-regulated,and it also found that a non-ribosomal peptide synthetase(NRPS)like protein was highly expressed.The DEGs analysis showed a high expression level of malate dehydrogenase(MDHC)and phosphoenolpyruvate carboxykinase(PCKA)in the CaCO_(3) group,which indicated a cytosolic malate activity.We speculated that the malate should be transported to or synthesized in the cytoplasm,which was then polymerized to PMLA by the NRPS-like protein,accompanied by the up-regulated TCA cycle providing ATP for the polymerization.Depending on the analysis,we assumed that an NRPS-like protein,the TCA cycle,and the cytosolic malate together are contributing to the PMLA biosynthesis.

Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.

短梗霉(Aureobasidium pullulans)与短梗霉多糖发酵

短梗霉多糖系短梗霉发酵产物,它具有巨大的经济价值。短梗霉是一种多形态真菌,具有复杂的生活史,并且其形态学变化与其多糖合成之间关系密切。本文就短梗霉及其发酵特点进行较详细的综述。

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Aureobasidium subglaciale F134 is a bifunctional whole-cell biocatalyst for Baeyer-Villiger oxidation or selective carbonyl reduction controllable by temperature

The microbial production of either ester/lactones or enantio-enriched alcohols through Baeyer-Villiger oxidation or stereoselective reduction of ketones,respectively,is possible by using whole cells of A.subglaciale F134 as a bifunctional biocatalyst.The chemoselective pattern of acetophenone biotransformation catalyzed by these cells can be regulated through reaction temperature,directing the reaction either towards oxidation or reduction products.The Baeyer–Villiger oxidation activity of A.subglaciale F134 whole cells is particularly dependent on reaction temperature.Acetophenone was transformed efficiently to phenol via the primary Baeyer–Villiger product phenyl acetate at 20℃ after 48 h with 100% conversion.In contrast,at 35℃,enantio-enriched(S)-1-phenylethanol was obtained as the sole product with 64% conversion and 89% ee.In addition,A.subglaciale F134 cells also catalyze the selective reduction of various structurally different aldehydes and ketones to alcohols with 40% to 100% yield,indicating broad substrate spectrum and good enantioselectivity in relevant cases.Our study provides a bifunctional biocatalyst systemthat can be used in Baeyer–Villiger oxidation aswell as in asymmetric carbonyl reduction,setting the stage for future work concerning the identification and isolation of the respective enzymes.

Studies on the optimal culture conditions of <i>Aureobasidium pullulans</i>to produce exopolysaccharides

In the current study, in order to change the permeability of cell membrane and solve the problem of linked group of fungi mycelium, the method of adjusting osmotic pressure of medium and adding tween-80 was established. The utilized strain with relatively high exopolysaccharide (EPS) yield and low pigment level was obtained after the rejuvenation and sifting of long-preserved Aureobasidium pullulans strain. The optimal proportion of substrate was determined by means of orthogonal test. The transformation ratio of EPS was increased by 10% - 20% and the pigment content was greatly reduced. The fermenting liquor is between creamy white and pale yellow, and the white primary product can be gained without decolourization step. Furthermore, to magnify to 5 L bioreactor can get the similar result.

The Synergism of Chemical Herbicides and Aureobasidium pullulans for Control Cleavers(Galium aparine L.) in Wheat

Aureobasidium pullulans, a biocontrol agent for the annual weed Galium aparine L. was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X （recommended field rate）, 0.5X, 0.2X, 0.1X 0.067X, and 0.05X concentrations. Germination of A. pullulans with paraquat, 2, 4-D, quizalofop-p, and ctethodim treatment appeared reduced compared with germination of A. pullulans with fluroxypyr treatment at all concentrations. Stunted and shorter germ tubes in comparison with the control were observed with 2, 4-D, quizalofop-p, and clethodim at 0.2X. All concentration of paraquat, 2, 4-D, quizalofop-p, and clethodim except 0.05X, significantly decreased radial growth of A. pullulans compared with its growth on the untreated PDA medium. Field trials to further develop A. pullulans as bio- control agent for control G. aparine L. was conducted to test the effectiveness of this fungus in wheat plots for 2 years at the same location in Xining. Treatments included spore suspensions of A. pullulans alone, a mixture of both fungus and fluroxypyr in wheat. Biocontrol agent effectiveness was estimated at approximately 7 and 14 days after treatment, as disease incidence, percent weed control, and weed biomass reduction. Significant reduction in weed biomass occurred in combination treatments, and potential exists to tank mix A. pullulans with fluroxypyr. Leaf surface moisture and air temperatures following application may account for inconsistencies in field results between years. This fungal organisms show potential as bioherbicides for weeds in G. aparine L.

出芽短梗霉Aureobasidium sp.SRF的菌株特性分析及胞外多糖结构鉴定

菌株SRF是1株从意大利树莓(Rubus corchorifolius)果实表面分离、可产胞外多糖的新菌株。在鉴定其分类归属的基础上,对其产生的胞外多糖进行了结构分析和发酵条件优化,为寻找微生物多糖提供新的菌株,为开发利用资源微生物提供借鉴。通过形态学和ITS序列对比分析进行菌株鉴定;通过薄层层析和红外光谱分析,确定胞外多糖结构;通过单因素检测试验,确定影响产糖量的主要因素;响应面Plackett-Burman和Box-Behnken设计筛选发酵产胞外多糖的最优条件。结果表明,出发菌株SRF隶属于出芽短梗霉属,命名为Aureobasidium sp.SRF;SRF所产胞外多糖为普鲁兰多糖;单因素检测表明,对多糖产量影响最大的因素为碳源浓度、氮源浓度、无机离子浓度,其次是碳源、氮源、无机离子、pH值;根据响应面结果确定最优发酵条件为麦芽糖8%(质量分数)、酵母提取物3%(质量分数)、钙离子0.3 g/L、pH 6,产糖量达5.93 g/L。SRF是1株来源于树莓浆果表面,可产胞外普鲁兰多糖的出芽短梗霉新菌株,是1株产微生物多糖的候选菌株。

深海出芽短梗霉(Aureobasidium sp.3A00493)菌株特征与胞外多糖特性分析

普鲁兰糖(pullulan)是短梗霉属(Aureobasidium spp.)的一些菌产生的极具高附加值、开发价值的天然微生物多糖。然而不同来源的菌株在产糖量、抑菌活性、副产物含量等方面有很大的差异,因此,寻找产糖量高、抑菌活性强、副产物少的优良菌株一直是研究人员关注的焦点。本研究的出发菌株3A00493是一株从太平洋深海沉积物中分离的产胞外多糖海洋微生物。通过形态学和显微镜观察、分子鉴定确定其分类归属;改变培养条件确定了其生长特性、最佳产糖条件;选择5种常见微生物作为指示菌,滤纸片法测定其抑菌活性;薄层层析和红外光谱分析了多糖结构。出发菌株3A00493是一株菌落乳白,细胞和菌丝体可转换生长,ITS和18S rDNA部分序列与报道短梗霉属(Aureobasidium sp.)同源性≥98%的海洋源出芽短梗霉菌;其最适生长温度为26℃,最适生长pH值为4,最适条件下产糖量达24.58 g/L;3A00493的代谢产物对4种指示菌具有较强抑菌活性;薄层层析和红外光谱测定结果表明3A00493产生的胞外多糖与普鲁兰糖标准品一致,确定为普鲁兰糖。出发菌株3A00493为一株产普鲁兰糖、代谢物抑菌活性较强、色素副产物少的海洋源出芽短梗霉新菌株。本研究为进一步研究海洋源产普鲁兰糖新菌3A00493奠定了理论基础,为寻找海洋源微生物多糖提供了新菌株,也为筛选和开发海洋资源微生物提供研究借鉴。

出芽短梗霉LHS-m022黑色素葡聚糖的发酵影响因素和生物活性

为探索出芽短梗霉LHS-m022黑色素多糖的发酵影响因素和生物活性,采用菌种发酵、分离提取及生物检测方法,对发酵液性质、产物收率、生物转化率及生物活性进行测定分析。结果表明,LHS-m022黑色素多糖发酵的最佳碳、氮源分别是蔗糖和胰酪蛋白胨,蔗糖最佳浓度为60 g/L;发酵培养基中诱导剂L-多巴的最适添加量为2.0 g/L,发酵液黑变活性和生物转化率分别是2.481和71.93%;细胞通透剂鼠李糖脂的最适添加量是0.021μL/L,发酵液黑变活性和生物转化率分别高达2.794和73.9%。全波长扫描、FTIR与HPLC分析表明,WAI为黑色素,PsB为黑色素葡聚糖结构。研究结果揭示,粗黑色素葡聚糖样品经121℃以上高温处理,仍然得到95.37%的絮凝率。采用1.50和2.00 g/L的样品浓度进行检测,分别得到99.55%的羟自由基清除活性和99.00%的抗氧化活性。

一株高产普鲁兰多糖短梗霉菌株的基因组分析及产糖机理探究

从分子水平解析微生物的基因组变化,从而对其进行遗传改造以提高普鲁兰多糖的产量是目前亟待解决的问题。通过比较基因组学方法,对一株高产普鲁兰多糖的出芽短梗霉(TN-31)与其他短梗霉菌株进行了基因组比较。结果显示,该菌株发生了基因组加倍,其基因组中存在大量的遗传变异,这可能是其高产普鲁兰多糖的重要原因。此外,该菌株的基因组大小高于其他短梗霉菌株,其特有基因和功能基因启动子区的特殊调控序列可能赋予其不同于其他短梗霉菌株的特性。这种特殊表型可能是适应性进化的结果,通过遗传变异和适应性选择,能够适应其所处的环境。

Aureobasidium pullulans UVMU3-1产耐热性胞外漆酶活性物质培养基优化及其对染料脱色作用

对出芽短梗霉无色素突变株Aureobasidium pullulans UVMU3-1产耐热性胞外漆酶活性物质培养基进行优化,并研究其分子量大小、热稳定性及对三苯甲烷类染料孔雀石绿和结晶紫的脱色作用.结果表明:蔗糖、NaNO_3为UVMU3-1产耐热性胞外漆酶活性物质的最佳碳源和氮源,产酶优化培养基配方为:蔗糖66.5 g·L^(-1),NaNO_338.4 g·L^(-1),NaCl 1.08 g·L^(-1),酵母浸粉0.2 g·L^(-1),KH_2PO_41 g·L^(-1),pH=6.0.影响UVMU3-1产耐热性胞外漆酶活性物质的因素依次为:起始pH>蔗糖>NaCl>NaNO_3,培养7 d时产生最大酶活(94.81 U·L^(-1)),比对照提高3.85倍.其分子量小于1000 D,具有良好的热稳定性,煮沸30 min后高温酶活仍残留72.50%.粗酶液(7.5 U·L^(-1))与三苯甲烷类代表性染料孔雀石绿和结晶紫75℃反应5 min后,脱色率分别为76.3%、77.5%,反应35 min后脱色率分别达到96.3%、95.3%.

产聚苹果酸出芽短梗霉的筛选、鉴定及发酵培养基优化

从自然土壤样本中筛选得到一株产聚苹果酸(PMLA)菌株,该菌株经形态学观察和分子进化分析鉴定为出芽短梗霉(Aureobasidium pullulans)。筛选菌株同时具有聚苹果酸、普鲁兰多糖和liamocin胞外聚合物的合成能力。通过单因素和响应面试验对该菌种产PMLA的发酵培养基组分进行优化,获得较优培养基组分为葡萄糖130 g/L、硝酸钠5 g/L、玉米浆1.22 g/L、氯化钾0.5 g/L、磷酸二氢钾0.1 g/L、七水硫酸镁0.2 g/L、碳酸钙30 g/L,采用该培养基获得聚苹果酸的最大产量为25.82 g/L,比优化前提高了70.72%。

基于出芽短梗霉发酵法制备β-聚苹果酸

通过改变出芽短梗霉Aureobasidium pullulans的培养与发酵的工艺条件,实现β-聚苹果酸的生成量和分子量的提高。试验表明,最佳培养条件和培养组分为:100 g/L葡萄糖,35 g/L蛋白胨,2 g/L丁二酸铵,0.1 g/L KH2PO4,0.3 g/L MgSO4·7H2O,0.5 g/L KCl, 0.05 g/L MgSO4,20 g/L CaCO3,0.5 g/L亮氨酸,pH值4.0,发酵温度25℃,培养时间6 d。在该条件下进行发酵培养,β-PMLA的产量提高到24.3 g/L,分子量提高到8 948 Da。

出芽短梗霉菌株PA-2胁迫下藜qRT-PCR内参基因的筛选与验证

为筛选适合出芽短梗霉菌株PA-2胁迫下藜中稳定表达的内参基因,在转录组数据(NCBI登录号为:SRA:SRR12674257)分析的基础上,以PA-2不同时间胁迫的藜叶片为材料,分析藜中ACT-1、TUB-1、GAPDH、eEF1α、eIF3、UBQ、PTB、COX10、UPL6、TIP41L、RAN-B1和IDH-5等12个内参基因的表达水平,并利用ΔCt、GeNorm、NormFinder、BestKeeper及RefFinder分析候选内参基因的表达稳定性。结果表明,内参基因COX10是PA-2不同时间胁迫下藜中最稳定的内参基因,最优组合是COX10+eEF1α。通过分析藜ACC基因的相对表达水平,验证了内参基因的可靠性,为今后杂草藜胁迫响应基因的表达分析提供了参考基因和理论依据。

短梗霉中聚苹果酸生物合成机制研究进展

聚苹果酸(PMA)是一种天然聚酯材料,具有优良的水溶性、生物相容性、非免疫原性等优点,在生物医药、吸水材料和食品工业等领域具有广泛应用前景。PMA主要通过短梗霉合成,本文介绍了短梗霉合成PMA的代谢途径、关键酶及代谢调控机制,并对PMA合成代谢研究作出展望。

Screening, purification, and characterization of an extracellular lipase from Aureobasidium pullulans isolated from stuffed buns steamers

An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions(30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide(DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca2+ and was slightly inhibited by Mn2+ and Zn2+ at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide(CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.

南极酵母菌珍珠发酵液护肤功效研究

以来源于南极和云南的出芽短梗霉菌株为研究对象,通过紫外辐射、低温和高渗透压力筛选,发现来源于南极的出芽短梗霉菌株NJ1P3具有极强的耐受极端环境的能力。由于珍珠在化妆品中的应用较为广泛,为了提高人体皮肤对珍珠的吸收率,将NJ1P3与珍珠粉进行联合发酵并且对其产物进行了抗氧化以及抵御UVA和UVB对细胞损伤的活性测试。结果显示,NJ1P3珍珠发酵液具有良好的抗氧化特性,保护细胞抵御UVA和UVB损伤的护肤功效。

基于Biolog FF技术解析辐射对产黑色素短梗霉代谢活性的影响

【目的】分析辐射对短梗霉代谢活性的影响,研究真菌耐辐射机理机制。【方法】分离鉴定一株产黑色素短梗霉,并进行菌株的^(60)Coγ射线照射试验。根据菌株的耐辐射能力,设置^(60)Coγ射线0、2500、5000和10000 Gy 4个辐射剂量,采用Biolog FF技术检测不同辐射下菌株对FF微平板中碳源的利用程度,分析辐射对产黑色素短梗霉代谢碳源活性的影响。【结果】不同辐射剂量下菌株AWCD值存在明显差异。随着辐射剂量的升高,细胞代谢活性明显下降,羧酸类和氨基酸的利用呈下降趋势,而碳水化合物的利用率呈上升趋势。随着辐射剂量增加,菌株对碳源的利用率发生了变化。在Biolog FF微平板95种碳源中,共有46个碳源被利用,其中7种碳源利用率随辐射剂量上升而增加。其中,D-核糖,蔗糖,D-阿拉伯糖,L-阿拉伯糖的利用率增加最为明显。【结论】辐射能明显降低产黑色素短梗霉的代谢活性,其在高剂量辐射下碳源利用类型会发生明显的改变,可能是该菌辐射应激和适应的一种有效机制。

出芽短梗霉致肺部真菌感染1例并文献回顾

出芽短梗霉是一种在自然界中普遍存在的暗色酵母样真菌,作为一种条件致病菌,在正常免疫力患者中感染极其罕见。本文报告一例免疫功能正常的年轻女性感染出芽短梗霉病例,该例患者免疫功能正常,临床表现不典型,行肺泡灌洗液宏基因组二代测序(mNGS)检查见出芽短梗霉生长,行伏立康唑抗真菌治疗后明显好转。通过文献回顾,发现全球报道亦少见,共31例患者,其中男性20例,女性11例,年龄4月龄~79岁,2例患者免疫功能正常,无感染高危因素,余有基础疾病、继发性免疫抑制、外伤、移植等病史。临床表现无特异性,主要与受累系统相关,经抗真菌治疗好转及痊愈24例,7例死亡。近来年真菌感染越来越常见,出芽短梗霉所致真菌感染可累及全身各个部位,无免疫缺陷机体亦可能感染。通过mNGS检查可有效检测病原菌,从而及时诊断,缩短病程,有效治疗。