脱乙酰氧基头孢菌素C合成酶/羟化酶基因cefEF的克隆及序列分析

利用氯化苄分别从真菌顶头孢(Cephalosporium acremonium)和产黄头孢(Acremonium chrysogenum)中提取总DNA,通过PCR方法扩增脱乙酰氧基头孢菌素C合成酶/羟化酶基因cefEF,结果只能从产黄头孢DNA中扩增出cefEF基因。测序结果表明,其与已报道的基因序列只有3个碱基的差异,推断的氨基酸序列只有2个氨基酸有差异,并未涉及活性中心。同时表明,国外所指的与该酶有关的顶头孢(Cephalosporium acremonium或Acremonium chrysogenum)对应的是国内的产黄头孢(Acremonium chrysogenum)。     

陕西红脂大小蠹天敌种类调查

对陕西红脂大小蠹Dendroctonus valens LeConte危害区的天敌种类调查表明,寄生于红脂大小蠹的病原真菌共有5种,其中幼虫期有头孢霉Cephalosporium sp.、球孢白僵菌Beauveria bassiana和拟卵孢霉Ovulariopsis sp.,成虫期有球孢白僵菌Beauveria bassiana、枝顶孢霉Acremonium sp.、头孢霉Cephalosporium sp.、木霉Trichoderma sp.4种,其中以球孢白僵菌和枝顶孢霉Acremonium sp.的致病能力最为显著。捕食性天敌昆虫主要有西岳蛇蛉Agulla xiyue Yang et Chou、日本弓背蚁Camponotus japionicus Mayr、中华红林蚁Formica sinensis Wheeler、蚁形郭公甲Thanasimus formicarius(L.)及纤细阎甲Platysoma attenuata(LeConte),它们对红脂大小蠹均有较明显的控制作用。寄生性天敌主要有1种寄生蝇和1种茧蜂。     

顶头孢霉pcbAB-pcbC双向启动子区域的克隆与应用

用PCR方法从丝状真菌顶头孢霉中克隆出全长 1 3kb的pcbAB_pcbC双向启动子DNA片段 ,通过转化子对博莱霉素的抗性证明了该启动子在顶头孢霉中的双向启动功能。另外 ,利用所克隆的pcbAB_pcbC双向启动子构建了一个用于顶头孢霉转化的质粒pYG13,并成功地将该质粒转化入顶头孢霉。pYG13含有博莱霉素抗性基因和透明颤菌血红蛋白基因 (vgb) ,Southern杂交和CO结合实验分析显示vgb整合到顶头孢霉的基因组DNA中并表达了有活性的透明颤菌血红蛋白。     

重组a-氨基酸酯水解酶合成头孢曲嗪

为了探索酶法合成头孢曲嗪的产业化工艺路线,从红纹黄单胞菌Xanthomonas rubrillineans中克隆-氨基酸酯水解酶基因全序列,转化入大肠杆菌中表达。以头孢曲嗪的合成转化率为指标,分别考察纯化的重组-氨基酸酯水解酶合成头孢曲嗪的最适温度、最适pH和最佳底物摩尔比。经聚丙烯酰胺凝胶电泳分析,重组-氨基酸酯水解酶的单体分子量为70 kDa。催化合成头孢曲嗪的最适pH为(6.0±0.1),最适温度为36℃。底物浓度约为7-ATTC 30 mmol/L、HPGM HCl 120 mmol/L,酶用量22 U/mL时,头孢曲嗪的转化率达到64.3%。结果为优化酶法合成头孢曲嗪的产业化工艺奠定了基础。     

一种产黄青霉突变体库的简单快速构建方法

产黄青霉(Pennicillium chrysogenum)是重要的工业丝状真菌,为更好地提高青霉素产量,了解青霉素合成的调控途径及相关基因的功能,突变体库的构建是一种有效途径。本研究以PCR扩增得到的T-DNA片段为外源DNA,通过PEG介导转化至产黄青霉原生质体中,成功地将含有外源博来霉素抗性基因(ble)和绿色荧光蛋白基因(gfp)的T-DNA插入到产黄青霉基因组中,构建了产黄青霉突变体库,并实现了ble基因和gfp基因在产黄青霉中的表达。该方法不依赖于农杆菌介导,无需构建二元表达载体,只需一步PCR即可实现外源基因的制备,简单快速,也为研究其它真菌基因功能和突变体库构建提供参考。     

高产王浆西蜂DNA分子中的相关基因标志筛选及其鉴定

为了探讨西蜂与高产王浆相关特异基因标记 ,用 12种随机引物 (P1～P12 )对产王浆量不同的4品系西蜂的基因组DNA进行了RAPD PCR分析 ,分别获得了产王浆量高、低不同西蜂的DNA多态性图谱 ,并从P2 引物的DNA多态性图谱中筛选出一差异DNA片段P2 316bp .将P2 316bp差异DNA片段用地高辛标记制备成探针 ,进行Southern杂交鉴定 .实验显示 ,探针与高产王浆西蜂基因组DNA的扩增产物出现了阳性杂交信号 ,而与低产王浆西蜂基因组DNA的扩增产物未出现阳性杂交信号 .结果表明 ,该差异性基因片段P2 316bp是西蜂高产王浆优良性状相关的遗传标记 ,序列为 30 5个核苷酸 .     

来源于Aspergillus candidus的乳糖酶基因的克隆及序列分析

从一株产乳糖酶的亮白曲霉（Aspergillus candidus)中克隆到了乳糖酶基因组DNA及cDNA序列（EMBL AC－CESSION No.AJ431643),序列分析表明,乳糖酶基因组DNA序列长3458bp,其中含有8个内含子,cDNA编码区长3015bp,共编码1005个氨基酸,前19个氨基酸为信号肽序列,氨基酸序列中共含有11个潜在的糖基化位点。将此基因在不同来源的乳糖酶基因序列进行比较发现,该基因与绝大多数乳糖酶基因同源性较低。虽与米曲霉ATCC20423的乳糖酶序列同源性较高,但其在酶学性质上更优于后者,亮白曲霉的乳糖酶基因可能是一个具有更广阔的生产应用前景的新基因。     

抗氯化汞真菌的分离和鉴定

从受汞污染的土壤中分离到一种抗汞真菌,经鉴定为烟草头孢霉(Cephalosporium tabacimum),并初步研究了F2号菌的某些特性。该菌不仅对汞化合物有较强的抗性,同时对其它多种重金属的抗性也较强。     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

【目的】克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性。【方法】利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构;通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性。【结果】从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh(GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸。序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axono-podis pv. citri str. 306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91%和83%,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str. GPEPC73的肽酶亲缘性最高。基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5°C以上的突变体。【结论】对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程。对高度柔性位点进行饱和突变的策略可以用于提高热稳定性。     

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase.

Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.     

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.     

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.     

The effect of thiols and the Ca2+ ionophore A23817 on growth and antibiotic production in Cephalosporium acremonium

Abstract Yeast-like blastosporic growth, lowered levels of isopenicillin N synthetase and deacetoxycephalosporin C synthetase/hydroxylase, and lowered antibiotic production were induced in Cephalosporium acremonium strain C.O. 728 by inclusion of cysteine or glutathione in the growth medium. These effects were similar to those observed under normal conditions with strain M 8650/4, which had higher levels of glutathione reductase and protein disulphide reductase than strain C.O. 728. The addition of cytochalasins A and B slightly increased yeast-like growth in C.O. 728, although levels of biosynthetic enzyme activity and antibiotic secretion were not significantly affected. The Ca²⁺ ionophore A23187 inhibited C.O. 728 growth, slowed penicillin secretion into the medium, and totally inhibited cephalosporin secretion. These results show that maintenance of Ca²⁺ levels and the absence of thiols are important for antibiotic production in Cephalosporium acremonium .     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum

A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in lambda EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB-pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase with a deduced Mr of 404,134. This enzyme contains three repeated domains and a consensus thioesterase active-site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37,469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB-pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.