粗糙脉孢菌（Neurospora crassa）木糖发酵的研究

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌（Neurospora crassa）AS 3.1602木糖发酵的影响。结果表明,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行,此时乙醇的转化率达到63.2%。木糖醇发酵适合在微好氧的条件下进行,转化率达到31.8%。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响,乙醇产生最适pH5.0,木糖醇产生最适pH4.0。在培养基pH为碱性条件时,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用,对乙醇和木糖醇的产生也有很大的影响。     

粗糙脉孢菌(Neurospora crassa)木糖发酵的研究

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌(Neurospora crassa)AS3．1602木糖发酵的影响。结果表明,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行,此时乙醇的转化率达到63．2％。木糖醇发酵适合在微好氧的条件下进行,转化率达到31．8％。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响,乙醇产生最适pH5．0,木糖醇产生最适pH4．0。在培养基pH为碱性条件时,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用,对乙醇和木糖醇的产生也有很大的影响。     

表观遗传学研究的模式生物——粗糙脉孢菌

丝状真菌粗糙脉孢菌是一种作为遗传学研究的经典模式生物.通过对粗糙脉孢菌5S r RNA基因的组成和在染色体上分布的研究,揭示了丝状真菌中存在的一种基因组防御机制——重复序列诱导的DNA点突变(RIP).通过对发生突变的5S r RNA假基因的研究还发现,粗糙脉孢菌中存在一种重要的表观遗传修饰——DNA甲基化,随后的深入研究使粗糙脉孢菌成为解析DNA甲基化机制的最重要模式生物之一.粗糙脉孢菌基因转化操作引起的营养生长阶段同源基因的沉默(quelling)是由RNAi途径调控的,同时该途径也是调控减数分裂过程中非配对DNA诱发的基因沉默(meiotic silencing)的关键.由于粗糙脉孢菌基因组简单,且存在与高等真核生物相同的DNA甲基化和多种组蛋白的修饰,使其成为今后深入研究组蛋白修饰与染色质重塑等表观遗传现象参与基因表达调控和基因组稳定性维持的重要模式生物之一.     

综述: 表观遗传学研究的模式生物——粗糙脉孢菌

丝状真菌粗糙脉孢菌是一种作为遗传学研究的经典模式生物．通过对粗糙脉孢菌5S rRNA基因的组成和在染色体上分布的研究,揭示了丝状真菌中存在的一种基因组防御机制——重复序列诱导的DNA点突变(RIP)．通过对发生突变的5S rRNA假基因的研究还发现,粗糙脉孢菌中存在一种重要的表观遗传修饰——DNA甲基化,随后的深入研究使粗糙脉孢菌成为解析DNA甲基化机制的最重要模式生物之一．粗糙脉孢菌基因转化操作引起的营养生长阶段同源基因的沉默(quelling)是由RNAi途径调控的,同时该途径也是调控减数分裂过程中非配对DNA诱发的基因沉默(meiotic silencing)的关键．由于粗糙脉孢菌基因组简单,且存在与高等真核生物相同的DNA甲基化和多种组蛋白的修饰,使其成为今后深入研究组蛋白修饰与染色质重塑等表观遗传现象参与基因表达调控和基因组稳定性维持的重要模式生物之一．     

粗糙脉孢菌中甾醇还原酶基因erg24对麦角甾醇合成的影响

粗糙脉孢菌为子囊菌中的高效纤维素降解菌,可以直接以纤维素为营养源进行生长。本研究以粗糙脉孢菌为实验对象,利用基因工程技术构建甾醇还原酶基因erg24的高表达菌株,分别以蔗糖、麦麸、玉米秸秆、小麦秸秆、杨树木屑、水稻秸秆6种物质的粉末为碳源培养野生型粗糙脉孢菌和erg24高表达菌株,利用半定量RT-PCR测定在不同培养条件下erg2、erg24和erg6 3个麦角甾醇合成相关基因的表达水平,采用HPLC方法测定不同培养条件下麦角甾醇的积累量。研究结果表明,分别以玉米秸秆、杨树木屑、水稻秸秆这3种粉末为碳源时,培养物中的erg2、erg24和erg6 3个基因表达量较高。在不同培养条件下erg24高表达菌株合成麦角甾醇量显著高于野生型粗糙脉孢菌的合成量,且以杨树木屑粉末为碳源培养时,所获得的麦角甾醇产量最高,为30.53μg/mg。结果表明erg24基因是粗糙脉孢菌合成麦角甾醇的关键基因之一,利用玉米秸秆、小麦秸秆、杨树木屑或水稻秸秆粉末为碳源培养粗糙脉孢菌时,可获得较高产量的麦角甾醇。研究结果为以农业废弃物为营养源,利用真菌生产麦角甾醇奠定了基础。     

Zymomonas mobilis工程菌的研究现状及应用

运动发酵单孢菌 (Zymomonasmobilis)由于其具有较高的乙醇产率、得率和耐乙醇能力等特性 ,因此在产乙醇方面日益受到人们广泛的关注。主要阐述了运动发酵单孢菌的来源及其基本特性 ,重点对基因工程技术构建运动发酵单孢菌 ,使其能够利用木糖生产乙醇方面的研究进展及应用前景进行了综述。     

全基因组水平扫描鉴定粗糙脉孢菌糖转运蛋白及其在酿酒酵母己糖发酵中的评价

木质纤维素降解真菌粗糙脉孢菌天然具有吸收利用多种单糖和寡糖的能力,但是目前基因组中注释的预测糖转运蛋白仍然有过半功能未知。本研究从全基因组水平系统分析了粗糙脉孢菌预测糖转运蛋白的转运底物。研究发现两个转运蛋白(NCU01868和NCU08152)具有转运多种己糖底物的功能,因此分别命名为NcHXT-1和NcHXT-2。利用荧光共振能量转移技术(FRET)确认了NcHXT-1/-2具有葡萄糖转运功能。在己糖转运蛋白全缺酿酒酵母EBY.VW4000中分别过表达NcHXT-1/-2,能恢复其在葡萄糖、半乳糖或甘露糖的液体培养基中生长并生成乙醇的能力。NcHXT-1/-2在很多纤维素降解真菌中均具有保守的同源蛋白。本研究通过全基因组扫描鉴定,发现了两个保守的丝状真菌己糖转运蛋白,为真菌降解利用木质纤维素及酵母利用单糖发酵提供了新的改造靶点。     

粗糙脉孢菌木质纤维素降解利用研究进展

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

粗糙脉孢菌木质纤维素降解利用研究进展简

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

一株粗糙脉孢菌的分离、鉴定及其基因组变异和功能分析

【背景】粗糙脉孢菌LY03是从武平传统"红菌豆腐"中分离得到的主要发酵菌株。【目的】研究粗糙脉孢菌LY03菌株的基因组信息,揭示武平传统"红菌豆腐"发酵特性。【方法】采用形态学观察、ITS鉴定、重测序及框架图测序对所分离的LY03菌株进行鉴定和基因组信息解析。【结果】武平传统"红菌豆腐"中分离得到的主要发酵菌株LY03确定为粗糙脉孢菌,将其在中国普通微生物菌种保藏管理中心进行专利保藏,保藏号为CGMCC3.19233。LY03菌株比对到参考基因组上的总Read数目为95.85%,测序对应深度的位点占全基因组76.13%;各变异类型在内含子区域均无变异,主要变异存在于基因组的外显子区域,具体变异数量为:单核苷酸多态性位点(single nucleotide polymorphism,SNP)位点变异总数203128个、插入缺失(insertion/deletion,In Del)突变总和26859个、拷贝数变异(copy number variation,CNV)增加和减少的拷贝总数1 039个、结构变异(structure variation,SV)注释的变异总数777个;LY03菌株...     

Production of ethanol from wood and agricultural residues by Neurospora crassa

The feasibility of utilizing the rapidly growing tropical woods for ethanol production by Neurospora crassa has been studied. Hydrolysis of cold alkali pretreated wood gave a saccharification of 68% based on the available carbohydrate. The direct fermentation of pretreated wood (20 g l^?1) by Neurospora crassa gave quantitative conversion of available hemicellulose/cellulose to ethanol in 5 days. Increasing the substrate concentration to 50 g l^?1lowered the conversion to 40–60% yielding 12 g l^?1of ethanol. Fermentation of wood (50 g l^?1) pretreated with hot 1 m NaOH followed by neutralization with HCl gave only 6 g l^?1of ethanol.     

Glucose Utilization and Ethanolic Fermentation by Wild Type and Extrachromosomal Mutants of Neurospora crassa

Logarithmic growth rates, maximal biomass, specific glucose utilization rates, and ethanol accumulation were measured in aerobic cultures of wild type and extrachromosomal mutants of Neurospora crassa. Maximal biomass and ethanol accumulation of wild type and [mi-1] were proportional to the initial glucose concentration in the range of 2 to 10%. The specific rates of glucose utilization by the mutants were 13- to 20-fold greater than those of wild type in young cultures. The specific rates of glucose utilization by wild type, however, were increased threefold by increasing the ammonium ion concentration in the preculture medium. The suppressor gene f(+) suppressed the excessive glucose utilization and enhanced the growth rate and maximal biomass of [mi-1]. When the mutants were utilizing glucose at excessive rates, ethanol did not appear in the culture medium. Ethanol accumulation was maximum at stationary phase or thereafter, but there was little difference between the maxima of the mutants and wild type. The molar efficiency of the conversion of glucose to ethanol during the entire culture period of wild type and mutants was about 50% and, in the latter stages of fermentation, approached 100%. Replacement of ammonium ion by nitrate in the culture medium suppressed ethanol accumulation by wild type. The relationship of these results to previous observations on respiratory adaptation are discussed. We suggest that the Pasteur effect, the inhibition of fermentation by respiration, may be operative in N. crassa. Factors such as nitrogen source and concentration and oxygen tension, which may serve primarily to regulate the amount and form of respiration would, therefore, indirectly regulate fermentation. The mutants, although transiently deficient in terminal respiratory activity, do not accumulate more ethanol than wild type and, therefore, apparently do not ferment in excess to obtain additional adenosine 5'-triphosphate. We suggest that the excess activity of the alternate form of respiration of the mutants may be related to their excessive rate of glucose utilization by way of the pentose phosphate pathway and the oxidation of excess reduced nicotinamide adenine dinucleotide.     

Cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain

We demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain expressing genes coding for cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) from Neurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.     

A novel biochemical route for fuels and chemicals production from cellulosic biomass

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.     

Lack of mutagenicity of vinyl chloride in two strains of Neurospora crassa.

No detectable induction of mutations could be found in two strains of Neurospora crassa after their conidia were treated with vinyl chloride, in ethanol solution and in its gaseous form. The results suggest that although N. crassa seems to lativating systems does not increase mutagenic activity of vinyl chloride in the two strains tested. At the same time these strains were mutated easily by UV and methyl methanesulfonate.     

Bioconversion of dilute-acid pretreated sorghum bagasse to ethanol by Neurospora crassa

Bioethanol production from sweet sorghum bagasse (SB), the lignocellulosic solid residue obtained after extraction of sugars from sorghum stalks, can further improve the energy yield of the crop. The aim of the present work was to evaluate a cost-efficient bioconversion of SB to ethanol at high solids loadings (16?% at pretreatment and 8?% at fermentation), low cellulase activities (1-7 FPU/g SB) and co-fermentation of hexoses and pentoses. The fungus Neurospora crassa DSM 1129 was used, which exhibits both depolymerase and co-fermentative ability, as well as mixed cultures with Saccharomyces cerevisiae 2541. A dilute-acid pretreatment (sulfuric acid 2?g/100?g SB; 210?°C; 10?min) was implemented, with high hemicellulose decomposition and low inhibitor formation. The bioconversion efficiency of N. crassa was superior to S. cerevisiae, while their mixed cultures had negative effect on ethanol production. Supplementing the in situ produced N. crassa cellulolytic system (1.0 FPU/g SB) with commercial cellulase and β-glucosidase mixture at low activity (6.0 FPU/g SB) increased ethanol production to 27.6?g/l or 84.7?% of theoretical yield (based on SB cellulose and hemicellulose sugar content). The combined dilute-acid pretreatment and bioconversion led to maximum cellulose and hemicellulose hydrolysis 73.3?% and 89.6?%, respectively.     

Immunopurification of highly specific antibodies to calmodulin from Neurospora crassa

Abstract Highly specific antibodies against calmodulin from Neurospora crassa were produced in rabbits. These antibodies were immunopurified by chromatography on Neurospora calmodulin-Sepharose, and had a titer for purified calmodulin from Neurospora crassa and bovine testis of 80 ng/ml and 2.5 μg/ml respectively. By immunoblot, as little as 8 ng of pure Neurospora calmodulin could be detected, and the antibodies revealed calmodulin in crude homogenates from Neurospora crassa and Aspergillus nidulans while in homogenates from Allomyces arbuscula, Saccharomyces cerevisiae, Dictyostelium discoideum and bovine testis, calmodulin remained undetected.