伪结核棒状杆菌感染影响巨噬细胞IL-1α表达、成熟及分泌的机制研究

【目的】伪结核棒状杆菌(Corynebacterium pseudotuberculosis,Cp)感染动物常引起内脏和体表淋巴结脓肿,以被感染部位炎性细胞因子大量增加为特征。研究Cp感染小鼠巨噬细胞对IL-1α成熟分泌的影响及机制。【方法】采用荧光定量PCR、ELISA和Western blotting等方法,首先检测Cp(ATCC19410、XH02)及其磷脂酶D基因(pld)缺失株(ATCC19410Δpld、XH02Δpld)感染巨噬细胞对IL-1α表达及成熟分泌的影响,进而以Ca²⁺螯合剂(EDTA、EGTA+Mg²⁺和BAPTA-AM)、calpain抑制剂(calpain inhibitor Ⅲ、calpain inhibitor Ⅳ和EST)处理巨噬细胞,观察对Cp感染介导IL-1α成熟分泌的影响。【结果】Cp感染巨噬细胞后,IL-1α mRNA表达及分泌显著增加,与ATCC19410、XH02感染巨噬细胞相比,ATCC19410Δpld、XH02Δpld感染细胞IL-1α表达及分泌显著下降。Cp感染引起巨噬细胞内Ca²⁺浓度显著增加,calpain活性增强,而缺失pld的Cp感染巨噬细胞内Ca²⁺浓度显著下降,calpain活性降低。EDTA、EGTA+Mg²⁺、BAPTA-AM、calpain inhibitor Ⅲ、calpain inhibitor Ⅳ处理Cp感染的巨噬细胞,巨噬细胞的IL-1α表达及分泌显著下降。【结论】Cp感染可激活巨噬细胞IL-1α表达和成熟分泌,磷脂酶D参与Cp感染巨噬细胞介导的IL-1α成熟分泌。Cp感染巨噬细胞介导IL-1α成熟与分泌与胞内Ca²⁺浓度增加、激活钙蛋白酶有关。     

基于CRISPR/Cas9系统筛选猪流行性腹泻病毒复制相关基因及其验证

【背景】成簇规律间隔的短回文重复序列相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas9)已被广泛证实是高效、强大的第三代基因编辑工具,在发现功能基因等领域取得了重要进展,但至今尚无利用该方法挖掘猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)宿主基因的报道。【目的】利用CRISPR/Cas9系统在全基因组范围内筛选PEDV复制相关基因,并进行候选基因的初步验证,为培育抗PEDV种猪提供科学参考。【方法】通过CRISPR/Cas9技术构建人肝癌细胞系(Huh-7)全基因组敲除文库,利用PEDV感染Huh-7文库细胞,随后经过高通量测序筛选影响PEDV复制的关键宿主因子,结合基因干扰和检测病毒效价等相关试验对影响PEDV复制的候选基因进行初步验证。【结果】构建了CRISPR/Cas9系统在全基因组范围内筛选PEDV复制相关基因的方法,将富集程度排名靠前的整合素α11(integrin α11,ITGA11)、哺乳动物复制蛋白A2(replication protein A2,RPA2)、驱动蛋白家族成员2A(kinesin family member 2A,KIF2A)、诱导髓系白血病细胞分化蛋白1(induced myeloid leukemia cell differentiation protein 1,MCL1)、多聚ADP核糖化酶1[poly(ADP-ribose)polymerase 1,PARP1]和囊泡单胺转运蛋白(vesicular monoamine transporter,SLC18A1)基因进行了验证;采用siRNA对上述基因分别进行干扰后,结果与对照组相比,干扰ITGA11可显著降低PEDV猪源靶向细胞IPEC-J2中PEDV-NmRNA、蛋白表达水平及子代病毒滴度。【结论】基于CRISPR/Cas9系统的全基因组敲除文库可作为挖掘PEDV复制相关功能基因的有效工具,ITGA11基因可作为一种制备抗PEDV猪种潜在的靶基因。     

敲除LTA4H基因的PK-15细胞系构建及其对口蹄疫病毒复制的影响

【目的】利用CRISPR/Cas9基因编辑技术构建白三烯A4水解酶(leukotriene A4 hydrolase, LTA4H)基因缺失的猪肾细胞系(porcine kidney-15, PK-15),探究LTA4H对口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制的影响,为开展LTA4H功能研究及调控病毒复制机制研究提供理论依据。【方法】设计2条针对猪LTA4H基因的引导RNA (small guide RNA, sgRNA),分别构建至载体pX459-puro-MCS中;将CRISPR重组质粒转染PK-15细胞,用嘌呤霉素(puromycin)抗生素筛选,并通过有限稀释法筛选单克隆细胞,之后通过Western blotting和测序检测LTA4H基因的敲除,获得LTA4H基因功能缺失细胞系。使用Western blotting、RT-qPCR及病毒滴度测定等方法检测敲除LTA4H基因后对FMDV复制及相关蛋白的表达情况。【结果】获得的单克隆敲除细胞系与野生型细胞相比,能够显著抑制FMDV复制。【结论】本研究成功构建了LTA4H基因敲除的PK-15细胞系,证明LTA4H对FMDV的复制具有促进作用,研究结果为后续LTA4H功能研究提供理论依据。     

CRISPR/Cas9介导的USP30基因敲除细胞系的建立及其对塞内卡病毒增殖的影响

【目的】利用规律成簇的间隔短回文重复序列/Cas9核酸酶（clustered regularly interspaced short palindromic repeats/Cas9 nuclease,CRISPR/Cas9）技术建立USP30基因敲除的人胚胎肾细胞（human embryonic kidney 293T cells,HEK-293T）细胞系,为开展宿主泛素特异性蛋白酶30（ubiquitin-specific protease 30,USP30）蛋白的功能研究建立了细胞模型;同时,初步探究USP30蛋白在病毒感染过程中的作用。【方法】根据Ensemble数据库查询USP30基因序列,定位USP30在基因组中不同转录本重叠区的第一个外显子段,设计并合成2对引导RNA （single guide RNAs,sgRNA）,分别构建在pX459载体中;将pX459-USP30-sgRNA质粒转染HEK-293T细胞,并用嘌呤霉素处理,筛选出转染阳性的细胞,然后通过有限稀释法筛选单克隆细胞,通过Western blotting及测序检测USP30基因的敲除。通过Western blotting及实时荧光定量PCR分析比较塞内卡病毒（Senecavirus A,SVA）在野生型和USP30基因敲除HEK-293T细胞中的复制差异。【结果】Western blotting及测序证实USP30基因敲除单克隆细胞系构建成功。进一步实验发现,SVA在USP30基因敲除细胞中的复制水平显著低于野生型细胞。【结论】成功构建USP30基因敲除的HEK-293T细胞系,首次证明USP30对SVA的复制具有促进作用,为进一步揭示USP30相关免疫反应和SVA感染过程的作用机制提供了良好的细胞模型,也为开展宿主USP30蛋白调控病毒复制的机制研究提供了关键工具和一定的理论依据。     

伪结核棒状杆菌pld基因无痕缺失株构建及其生物学特性及致病性研究

伪结核棒状杆菌是一种可以感染多种动物和人的兼性胞内寄生病原,主要引起被感染动物慢性化脓性炎症反应。[目的]为进一步评价磷脂酶D基因(pld)在伪结核棒状杆菌感染致病中的作用。[方法]本研究采用同源重组技术,在不引入外源基因情况下,构建伪结核棒状杆菌pld无痕缺失株。通过比较pld缺失株和野生株的菌落形态及生长曲线、体外对巨噬细胞乳酸脱氢酶(LDH)释放及其在胞内的繁殖情况以及体内感染小鼠致死率及促炎细胞因子分泌水平,研究pld与该病原感染致病之间的关系。[结果]无痕缺失pld对伪结核棒状杆菌的菌落形态及生长无明显影响。与野生株ATCC 19410和XH02相比,ATCC 19410Δpld和XH02Δpld失去了与马红球菌ATCC 6939的协同溶血功能,所感染巨噬细胞的LDH释放水平显著下降,胞内细菌数量显著降低;体内试验发现ATCC 19410Δpld对小鼠的致死率、肝脏和脾脏载菌量以及腹水及脏器促炎细胞因子水平均低于ATCC 19410。[结论]成功构建了伪结核棒状杆菌pld无痕缺失株。证实pld在该病原体外感染引发巨噬细胞死亡以及体内感染小鼠致病中具有重要作用。     

一种新型的CRISPR/Cas9-hLacI双链DNA供体适配基因编辑系统

在CRISPR/Cas9系统介导的基因编辑中,借助于双链DNA （double-stranded DNA,dsDNA）供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入,然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率,本研究利用大肠杆菌（Escherichia coli）乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点,通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌（Streptococcus pyogenes）源的SpCas9和路邓葡萄球菌（Staphylococcus lugdunensis）源的SlugCas9-HF融合表达,通过PCR将操纵序列LacO与dsDNA供体嵌合,构建了新型的CRISPR/Cas9-hLacI供体适配系统（donor adapting system,DAS）。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复（homology-directed repair,HDR）效率进行了验证和优化,其次在基因组水平对其介导的基因精确编辑进行了检测,并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑,效率高达30.5%,显著高于野生型。综上所述,本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统,丰富了CRISPR/Cas9基因编辑技术种类,为以后的基因编辑及分子设计育种研究提供了新的工具。     

圆红冬孢酵母自主复制序列分离和利用游离型质粒进行基因敲除

【目的】与整合型表达载体相比,游离型表达载体通常具有更高的拷贝数以实现目标基因的高强度表达,并且对于DNA操作应用更加方便和灵活。然而,目前的研究尚未确定适用于圆红冬孢酵母的游离型质粒,该酵母外源基因的表达或者基于CRISPR/Cas9的基因组编辑都需要通过整合方式来完成,这也是对其遗传改造进展缓慢的一个重要原因。本研究目的是构建圆红冬孢酵母的游离型质粒,使得其外源基因的表达和基因组编辑更方便省时。【方法】首先对圆红冬孢酵母苯丙氨酸氨裂解酶基因(phenylalanine ammonia-lyase gene, PAL)中可能存在的自主复制序列(autonomously replicating sequences, ARSs)进行挖掘和表征,将该基因及其上下游序列进行分段扩增,构建到带有β-异丙基苹果酸脱氢酶基因(β-isopropyl malate dehydrogenase gene, LEU2)的质粒中,通过电转化的方法导入LEU2基因缺陷的圆红冬孢酵母中,根据转化效率高低鉴定了该酵母的一个ARS。其次,以编码香叶基香叶基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase, GGPPS)的BTS1基因为敲除靶点,将其gRNA构建到基于ARS的游离型质粒中,通过转化子直观的颜色变化来验证该游离型质粒是否成功应用于圆红冬孢酵母的CRISPR/Cas9体系。【结果】本工作鉴定了圆红冬孢酵母的ARS,构建了基于ARS元件的游离型质粒,并将该质粒应用于圆红冬孢酵母CRISPR/Cas9体系,成功实现了基于游离型质粒的基因敲除。【结论】本研究丰富了圆红冬孢酵母现有的工具库,为圆红冬孢酵母的合成生物学应用提供了良好的研究基础和技术支持。     

拟态弧菌金属蛋白酶基因PrtV缺失株的构建及生物学特性分析

【背景】研究发现PrtV基因编码含多囊肾病(polycystic kidney disease)结构域的金属蛋白酶,其在多种细菌的致病过程中具有重要作用。拟态弧菌是一种感染多种水生动物的重要病原菌,PrtV基因在拟态弧菌致病中的作用尚不清楚。【目的】探究PrtV基因对拟态弧菌致病相关生物学特性的影响。【方法】采用自然转化的方法构建拟态弧菌PrtV基因缺失株(ΔPrtV),同时通过基因与质粒重组后电转化导入缺失株构建回补株(ΔPrtV/pPrtV),对突变株的生长特性、生化特征、生物被膜形成、自聚集能力、胞外产物卵磷脂酶和蛋白酶活性,以及致病性和细胞毒性等进行分析。【结果】与野生株相比,缺失株的生长特性、生物被膜形成、自聚集能力和卵磷脂酶活性无变化,但分解尿素、甘氨酸、香豆酸盐、鸟氨酸和赖氨酸的理化特性改变;胞外产物蛋白酶活性显著降低(P<0.05),细胞毒性显著下降(P<0.05),对杂交鲇的致病力下降10倍。【结论】PrtV基因与拟态弧菌的细胞毒性及致病性等多种生物学特性有关。该结果为进一步解析拟态弧菌PrtV基因功能及其致病机制提供了依据。     

绣球‘杜丽''AP3基因克隆与基因编辑载体构建

绣球(Hydrangea macrophylla)是以花序为主要观赏部位的园林植物,多用作切花装饰和景观营造,在亚洲、美洲、欧洲广泛栽培。为探究AP3基因在绣球花萼形成过程中的功能,加快重瓣绣球新品种培育进程,该研究以绣球‘杜丽''为材料,克隆其MADS-box B类基因HmAP3,并结合生物信息学方法预测基因功能; 根据HmAP3序列信息,筛选出高特异性编辑靶点并构建CRISPR/Cas9基因编辑载体,通过农杆菌转化法将载体整合到绣球基因组中。结果表明:(1)克隆到1段HmAP3基因的cDNA序列,其序列全长546 bp,共编码181个氨基酸,测序结果表明其氨基酸序列与参考序列一致性为100%,与拟南芥AtAP3相似度为58.8%。(2)不同属植物AP3氨基酸序列差异较大,在同属不同物种中,AP3蛋白主要结构较为保守,仅在少数基序上存在差异。(3)在HmAP3中共鉴定到2个高特异性靶点,并成功构建2个单靶点CRISPR/Cas9基因编辑载体。(4)该研究共获得5株基因组内含有Cas9序列的抗性芽,但其靶点均未突变,在抗性芽中没有检测到Cas9表达。该研究探讨了AP3基因在重瓣绣球育种中的价值,对绣球的CRISPR/Cas9基因编辑技术进行了初探,为绣球优良品种繁育工作奠定了基础。     

一种高效无选择标记的黑曲霉基因组编辑方法

黑曲霉(Aspergillus niger)是一种重要的工业生产菌株,被广泛地应用于生产酶制剂和有机酸,但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术,但由于需要在基因组中整合选择标记或基因编辑效率还有待提高,影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先,利用5S rRNA启动子启动sgRNA的表达,构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒;同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株;最后利用含有AMA1片段质粒的不稳定性,通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法,在采用同源臂长度仅为20bp的无选择标记供体DNA进行基因编辑时,基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.