The cel4 Gene of Agaricus bisporus Encodes a β-Mannanase

Mannases have industrial uses in food and pulp industries, and their regulation may influence development of the mushrooms of commercially important basidiomycetes. We expressed an Agaricus bisporus cel4 cDNA, which encodes a mannanase, in Saccharomyces cerevisiae and Pichia pastoris. CEL4 had no detectable activity on cellulose or xylan. This gene is the first isolated from this economically important fungus to encode a mannanase. P. pastoris secreted about three times more CEL4 than S. cerevisiae. The removal of the cellulose-binding domain of CEL4 lowered the secreted specific activity by P. pastoris by approximately 97%. The genomic sequence of cel4 was isolated by screening a cosmid library of A. bisporus C54-carb8. The open reading frame was interrupted by 12 introns. The level of extracellular CEL4 increases dramatically at the postharvest stage in compost extracts of A. bisporus fruiting cultures. In laboratory liquid cultures of A. bisporus, the activity of CEL4 detected in the culture filtrate reached a maximum after 21 days. The levels of CEL4 broadly mirrored the levels of enzyme activity. In the Solka floc-bound mycelium, CEL4 protein showed a maximum after 2 to 3 weeks of culture and then declined. Changes in CEL4 activity during fruiting-body development suggest that hemicellulose utilization plays an important role in sporophore formation. The availability of the cloned gene will further studies of compost decomposition and the extracellular enzymes that fungi deploy in this process.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.     

CEL1: a novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose

Abstract The cell gene of Agaricus bisporus encodes a protein (CEL1) that has an architecture resembling the multi-domain fungal cellulases, although the sequence of its putative catalytic core is not matched by any other in the protein and nucleic acid data bases. The N-terminal half of the putative catalytic domain of CEL1 was expressed in Escherichia coli as a fusion protein with glutathione- S -transferase. The fusion protein was used to raise a CEL1-specific antibody. CEL1 was detected as an extracellular 49.8 kDa protein in A. bisporus cellulose-grown cultures, where it bound strongly to cellulose. CEL1 was neither an endoglucanase, a cellobiohydrolase able to hydrolyze fluorogenic cellobiosides, a β-glucosidase, a xylanase, nor a cellobiose: quinone oxidoreductase. CEL1 was present in some fractions of culture fluid separated by electrophoresis which released soluble sugars from crystalline cellulose.     

耐碱性甘露聚糖酶基因的克隆及其在毕赤酵母中的表达

通过功能平板从土壤中筛选得到含甘露聚糖酶基因的耐碱菌株。构建其基因组文库,从中筛选到甘露聚糖酶基因TM1并测序分析,用BLAST分析表明,TM1的氨基酸序列与其他在GenBank发表的甘露聚糖酶的氨基酸序列的同源性均低于60%,故确定其为一个新的甘露聚糖酶基因(GenBank登录号为AY623903)。将此基因去除信号肽后的编码序列克隆到表达载体pHBM905C上,得到重组质粒pHBM1201。经SalⅠ酶切后分别转化毕赤酵母(Pichiapastoris)KM71、GS115、SMD1168,得到分泌表达的重组毕赤酵母。挑选相对表达量最高的重组毕赤酵母SMD1168-3在摇瓶中诱导产酶,对该酶的粗酶进行酶学性质分析表明,其最适反应温度为55℃,最适PH值为7.5,以魔芋粉为底物所测得的最高酶活为41.8U,半衰期为1h,在80℃保温5min其酶活由最初酶活的77%下降到11%,温度下降到55℃后活性可恢复到最初酶活的60%以上。     

Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

We used the yeast MEL1 gene for secreted alpha-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi beta-1,4-exoglucanase was inserted into one cartridge to create a fusion of the alpha-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-beta-d-cellobiose, and p-nitrophenyl-beta-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K(m) and pH optimum but to be more thermostable.     

猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ分泌型重组表达载体, 电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDS-PAGE和Western blot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,其表达量为108mg/L,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFN-γ基因的分泌表达。     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P. pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P. pastoris GS115, 1176 U mg(-1) protein by P. pastoris KM71H and 1522 U mg(-1) protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.     

Molecular cloning and functional expression of a novel extracellular lipase from the thermotolerant yeast Candida thermophila

The thermotolerant yeast Candida thermophila SRY-09 isolated from Thailand produces an extracellular lipase that hydrolyses various triglycerides. To clone the gene encoding the lipase, Saccharomyces cerevisiae was transformed with a C. thermophila genomic library and screened for lipase activity on medium containing olive oil emulsion and rhodamine B. One C. thermophila lipase gene (CtLIP) was found that contained an ORF of 1317 bp encoding a deduced polypeptide of 438 amino acids. Candida thermophila lipase contained a Gly-Asp-Ser-Gln-Gly motif which matched the consensus Gly-X-Ser-X-Gly conserved among lipolytic enzymes. Heterologous expression of the cloned CtLIP under the control of the alcohol oxidase gene (AOX1) promoter in the methylotrophic yeast Pichia pastoris, and enzymatic measurements confirmed the function of the respective protein as a lipase. The recombinant CtLIP could hydrolyse various substrates at high temperature (55 degrees C) with higher efficiency than at 37 or 45 degrees C and preferentially hydrolysed two-positional ester bonds. As with C. thermophila, the heterologously expressed lipase was secreted into the medium by Pichia pastoris.     

Cloning,expression and characterization of endo‐β‐1,4‐mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo‐β‐1,4‐mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA ) and the alcohol oxidase (AOX1 ) promoters, respectively. The highest production of mannanase (352 U mL^?1) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL^?1). The fold increase in mannanase production was estimated as ～12‐fold and ～2‐fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (～350 U mg^?1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

里氏木霉双基因同步缺失菌株的构建与甘露聚糖酶表达研究

在里氏木霉中建立了一个快速的双基因位点同步同源重组新方法,较好解决了里氏木霉基因逐个敲除周期长等问题。研究以里氏木霉自身甘露聚糖酶基因（man5A）为重组表达的报告基因,通过一步转化,将该基因定点整合入纤维二糖水解酶Ⅰ（cbh1）基因位点,同时缺失主要的两个纤维素酶基因（cbh1、cbh2）,得到重组工程菌Man12。将重组工程菌Man12与出发菌株Tu6Δku70进行摇瓶发酵,结果显示,重组菌株的甘露聚糖酶产量比出发菌株提高10倍,而纤维素酶产量降低了60%,胞外总蛋白分泌水平降低了40%。Real-time PCR检测甘露聚糖酶基因（man5A）的转录水平,发现重组菌株较出发菌株提高了25倍。在里氏木霉中首次报道了通过一步转化实现两个基因同步定点整合的方法,对利用基因工程手段构建高效表达重组蛋白的里氏木霉工程菌株具有一定的指导意义。     

植酸酶和甘露聚糖酶双功能毕赤酵母工程菌的构建和产酶分析

根据已发表的植酸酶基因和甘露聚糖酶基因序列设计并合成引物,应用PCR技术,分别以土曲霉总DNA和质粒pHBM1201为模板,扩增出均不含假定信号肽序列的植酸酶基因phyA和甘露聚糖酶基因man,将它们各自克隆到毕赤酵母表达载体pHBM907C上,分别得到重组质粒pHBM907C-phyA和pHBM907C-man。将质粒pHBM907C-phyA上由乙醇氧化酶(AOX1)启动子和终止子引导表达、酿酒酵母α信号肽序列引导分泌的phyA表达盒式结构插入到质粒pHBM907C-man中,构成双基因表达分泌质粒pHBM907C-phyA-man。pHBM907C-phyA-man经SalⅠ酶切线性后转化毕赤酵母(Pichiapastoris)GS115,获得了同时分泌表达植酸酶和甘露聚糖酶的双功能酵母工程菌。研究了该酵母工程菌所分泌表达的重组植酸酶和甘露聚糖酶的相关酶学性质,并进行了双功能酵母工程菌的稳定性测试。     

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris

A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Saprotrophic and mycoparasitic components of aggressiveness of Trichoderma harzianum groups toward the commercial mushroom Agaricus bisporus

We examined the mycoparasitic and saprotrophic behavior of isolates representing groups of Trichoderma harzianum to establish a mechanism for the aggressiveness towards Agaricus bisporus in infested commercial compost. Mycoparasitic structures were infrequently observed in interaction zones on various media, including compost, with cryoscanning electron microscopy. T. harzianum grows prolifically in compost in the absence or presence of A. bisporus, and the aggressive European (Th2) and North American (Th4) isolates produced significantly higher biomasses (6.8- and 7.5-fold, respectively) in compost than did nonaggressive, group 1 isolates. All groups secreted depolymerases that could attack the cell walls of A. bisporus and of wheat straw, and some were linked to aggressiveness. Growth on mushroom cell walls in vitro resulted in rapid production of chymoelastase and trypsin-like proteases by only the Th2 and Th4 isolates. These isolates also produced a dominant protease isoform (pI 6.22) and additional chitinase isoforms. On wheat straw, Th4 produced distinct isoforms of cellulase and laminarinase, but there was no consistent association between levels or isoforms of depolymerases and aggressiveness. Th3's distinctive profiles confirmed its reclassification as Trichoderma atroviride. Proteases and glycanases were detected for the first time in sterilized compost colonized by T. harzianum. Xylanase dominated, and some isoforms were unique to compost, as were some laminarinases. We hypothesize that aggressiveness results from competition, antagonism, or parasitism but only as a component of, or following, extensive saprotrophic growth involving degradation of wheat straw cell walls.     

Production of \-mannanases by Trichoderma harzianum E58

Summary The extracellular mannanase and endoglucanase activities of Trichoderma harzianum E58 were followed during growth of the fungus on 1% (w/v) mannose, Avicel, locust bean gum, konjac powder or the water-soluble fraction from stream-treated white spruce (SWS). Peak galactomannanase activities of 0.60 IU/ml and 0.66 IU/ml were detected in culture filtrates after 6–8 days growth on locust bean gum and Avicel respectively. When SWS or konjac powder were used as substrates, lower but relatively constant levels of activity were detected between 2 and 11 days of growth. Growth of the fungus on mannan-rich locust bean gum resulted in the highest specific glucomannanase and galactomannanase values. Although growth on 1% mannose failed to induce any mannanase activity, when 0.5% galactomannan was added with mannose, mannanase activity was detected in the culture filtrate. This indicated that mannanase production was not repressed in the presence of mannose. Samples were taken from each culture at the time of maximum galactomannanase activity. A protein profile obtained by isoelectric focusing was followed by a zymogram overlay to detect bands exhibiting galactomannanase, glucomannanase and endoglucanase activities. Several bands showed mannanase and endoglucananase activity. One band at pI 6.55 revealed both gluco- and galactomannanase activity and was free of detectable cellulase activity. Offprint requests to: J. N. Saddler     

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).     

Effect of spent mushroom compost tea on mycelial growth and yield of button mushroom (Agaricus bisporus)

Preliminary studies suggested that the use of compost tea made from spent mushroom substrate (SMS) may be regarded as a potential method for biologically controlling dry bubble disease in button mushroom. The aim of this study was to assess the effect of SMS compost tea on the host, the button mushroom, to ascertain whether the addition of these water extracts has a toxic effect on Agaricus bisporus mycelium growth and on mushroom yield. In vitro experiments showed that the addition of SMS compost tea to the culture medium inoculated with a mushroom spawn grain did not have an inhibitory effect on A. bisporus mycelial growth. The effect of compost teas on the quantitative production parameters of A. bisporus (yield, unitary weight, biological efficiency and earliness) was tested in a cropping trial, applying the compost teas to the casing in three different drench applications. Quantitative production parameters were not significantly affected by the compost tea treatments although there was a slight delay of 0.8-1.4 days in the harvest time of the first flush. These results suggest that compost teas have no fungitoxic effect on A. bisporus so that they can be considered a suitable biocontrol substance for the control of dry bubble disease.     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009