Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

牛源近平滑念珠菌对氟康唑耐药性研究

以牛源近平滑念珠菌（Candida parapsilosis）为试验菌株,采用微量稀释法进行药物敏感性试验,PCR扩增测序检测ERG11基因突变,Realtime PCR检测ERG11、CDR1、MDR1、MRR1基因的mRNA表达量,探讨耐药相关基因在牛源近平滑念珠菌耐唑类药物中的作用,为牛源近平滑念珠菌的耐药研究提供参考。结果表明,近平滑念珠菌对5-氟胞嘧啶、两性霉素Ｂ的敏感率均高于75%,对唑类药物的耐药率均高于50%,其中对氟康唑的耐药率最高,达58.3%;所有菌株的ERG11基因中均检测出错义突变A395T,耐氟康唑和剂量依赖菌株的ERG11基因中检测出同义突变T591C;氟康唑耐药组ERG11、CDR1、 MDR1、MRR1基因表达水平均显著高于敏感组（P<0.05）。牛源近平滑念珠菌对唑类抗真菌药物的耐药率较高且具有多重耐药性。牛源近平滑念珠菌ERG11基因中的T591C突变以及ERG11、CDR1、MDR1、MRR1基因的高表达都可能在其对氟康唑耐药性的产生中起到一定的作用。     

Expression and immunolocalization of ERG1 potassium channels in the rat kidney

Potassium (K⁺) channels participate in K⁺ secretion, K⁺ recycling, and cell volume regulation and help to maintain the resting potential in mammalian kidneys. Previously, we identified a set of voltage-gated K⁺ channels (Kv1) in the inner medullary collecting duct of the rat kidney. In the present work, we identified the voltage-gated K⁺ channel ether-à-go-go-related gene (ERG) in the rat kidney. mRNAs of ERG1a and its N-terminal splice-variant ERG1b were detected. Immunoblots of the cortex and medulla revealed two molecular mass proteins of 135 and 80 kDa, consistent in size with the nonglycosylated ERG1a and ERG1b isoforms, respectively. However, bands of 155 and 95 kDa, corresponding to mature glycosylated ERG1a and ERG1b, respectively, were also observed. In our immunohistochemical experiments, we could not differentiate the ERG1 isoforms because we used an antibody against a carboxy-terminal epitope. ERG1 was differentially localized in specific nephron segments: its localization was intracellular in the proximal tubule and medullary collecting ducts and in the apical membranes in the distal convoluted and connecting tubules. ERG1 was also abundant in glomerular arterioles and renal vessels. In summary, ERG1 displays a heterogeneous distribution in the rat kidney.     

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml⁻¹. In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml⁻¹. The results obtained from real-time PCR and analysis of the ∆∆C_q values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.     

TheRT1.G locus in the rat encodes a Qa/TL-like antigen

A new antigenic system in the rat homologous to theQa/TL antigen system in the mouse has been characterized. It was detected by antibodies raised in donor-recipient combinations that were matched for theRT1. A, B, D, E loci in the major histocompatibility complex (MHC): (R11×BN)F₁ anti-BN.1L(LEW), (R18×BN)F₁ anti-BN.1L, and BN.1LV1(F344) anti-BN.1L. Absorption analyses using these antisera and a variety of inbred, congenic and recombinant strains identified three alleles,RT1.G ^a ,G ^b ,G ^c , of whichG ^c is a null allele. The strain distribution of these alleles was determined, using 37 strains of rats representative of all of the prototypic haplotypes and a number of congenic and recombinant strains. The use of the congenic and recombinant strains showed that theRT1.G locus was linked to the MHC and that the most probable gene order wasA-E-G. Testcross analysis showed that the map distance betweenA andG was 1.4 cM(4/285 recombinants). The RT1.G antigen has a heavy chain ofM _r 46 000 and is present on both T and B cells.     

Partial exclusion of genes of bacteriophage T2 with T4-glucosylated DNA in crosses with bacteriophage T4

A recombinant strain (D41) between phage T2 and T4 was isolated which possessed the T2 region of the genome between genes 32 and 39 and both the T4 genesgt ⁺ andgt ⁺ for glucosyltransferase. D41 was crossed with T4amber mutants in the genes for early functions and in some genes for late funcitions. The progeny of the crosses was examined for the frequency of theam ⁺ markers from D41. Genes 32, 60 and 39 in the T2 region of the recombinant strain were as sensitive to exclusion as those in standard-type T2. The T4 glucosylation of the DNA of these T2 genes did not protect them against partial exclusion by T4. However, genes in the region from gene 56 to 55 in the recombinant were resistent to exclusion. In standard-type T2 this region of the genome is sensitive to partial exclusion by T4. There are at least four exclusion sensitive sites in T2: one near gene 32, one near gene 60, one linked to gene 56 and one between genes 42 and 55.This investigation was carried out partially within the frame of the Association between Euratom and the University of Leiden, contract nr. 052-64-1-BIAN.     

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10^?8/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10^?8/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

High expression of Trigonopsis variabilis -amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of -amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his⁻mut⁺). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his⁺mut⁺) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.     

Correlation between cell lipid content, gene expression and fermentative behaviour of two Saccharomyces cerevisiae wine strains

Aim: To verify a possible correlation between cell lipid composition, expression of key genes in lipid metabolism and fermentative behaviour of Saccharomyces cerevisiae wine strains. Methods and Results: The fermentative abilities of two commercial wine strains of S. cerevisiae were tested under stressful conditions. Cell number, glucose and fructose concentrations, expression of ACS1, ACS2, ACC1, OLE1, ERG9, ERG10, ARE1 and ARE2 and lipid content were evaluated. The strain that failed to complete the fermentation had lower amounts of C16:1 and C16:0 fatty acids at the beginning of fermentation (0 h) and late logarithmic phase (72 h). While the amount of C18:1 in this strain was lower than that in the strain that completed the fermentation at 0 h, same levels were observed for both strains at 72 h. The sterol levels were generally higher in the strain that failed to complete the fermentation. Gene expression generally increased from the beginning of the fermentation to the late logarithmic phase in both strains. Conclusion: A positive correlation between good fermentative ability, elevated fatty acid content and ACC1 gene expression has been identified. Significance and Impact of the Study: The cell lipid content at the time of inoculum and expression of ACC1 gene of starter strains should be carefully considered in order to identify the possible stuck/sluggish fermentations.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999     

Evaluation of strains derived from Escherichia coli W as hosts for the expression of penicillin G-acylase-encoding gene cloned on the recombinant plasmid pKA18

The potential for production of penicillin G-acylase (PGA), encoded by the chromosomal genepga ⁱ, of four strains belonging to a genealogical line derived from the strainEscherichia coli W, was evaluated in a medium with and without the inducer phenylacetic acid (PA). These strains were used as hosts of the recombinant plasmid pKA18, in which the structural genepga ^c isolated from the strain RE3, the best host strain of a line giving the highest production, was cloned. The presence of the inducer reduced the copy number of the plasmid in all recombinant strains. Only in recombinant strain RE3 (pKA18) the reduction of the gene dosage ofpga ^c resulted also in the reduction of the amount of PGA synthesized by the cells. The reduced activity of the cells did not result from a segregation of plasmid-free clones. Also the growth rate was decreased by 20 and 40% in the host and recombinant strains, respectively. The host strain RE3 showing the highest production of PGA was also the best host of the recombinant plasmid in terms of the segregational stability and copy number (198 copies per chromosome). The recombinant strain RE3 (pKA18) also provided the highest production of PGA.     

甾醇C-22去饱和酶高表达对酵母细胞麦角甾醇合成的影响

通过PCR扩增克隆到酵母菌甾醇C-22去饱和酶基因(ERG5)的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pYPE5。以铜离子螯合蛋白基因CUP1替换ERG5基因内部序列获得ERG5破坏菌株YSE5,其中麦角甾醇的合成被阻断,而积累了甾醇中间体Ergosta-5,7-dien-3β-ol。表达质粒pYPE5转化破坏菌株后使细胞恢复了合成麦角甾醇的能力。说明表达质粒上的ERG5基因得到了功能性的表达。将表达质粒pYPE5转化酿酒酵母单倍体菌株YS58,通过营养缺陷互补筛选到重组菌株YS58(pYPE5)。对重组菌株、破坏菌株和互补菌株细胞甾醇组分和含量进行测定,发现重组菌株和互补菌株的麦角甾醇和总甾醇含量明显低于对照菌YS58(YEp352)。测定不同培养时间细胞的麦角甾醇含量,发现重组菌株的麦角甾醇含量始终低于对照菌YS58(YEp352)。可见,ERG5在酵母中的高表达导致细胞麦角甾醇含量降低。     

Expression of alkaline proteinase gene in two recombinant <Emphasis Type="Italic">Bacillus cereus</Emphasis> feather-degrading strains

Two Bacillus cereus feather-degrading strains (23/1 and 6/2) were transformed using a recombinant plasmid p5.2 carrying the alkaline proteinase gene (aprE). A high level of the aprE gene expression was observed when the recombinant strains were grown on sporulation medium. The expression of the aprE gene proceeded during the early stationary phase and the p5.2 plasmid was segregationally and structurally stable in both strains. The two recombinant strains grown on a mineral medium with 1 % chicken feather as source of energy, carbon and nitrogen exhibited higher proteolytic activity (≈6-fold and 2.4-fold higher for strains 23/1 (p5.2) and 6/2 (p5.2), respectively. Keratinolytic activity increased ≈3.5-fold and 4.15-fold, respectively. The keratinolytic activity further increased when an optimized medium with yeast extract and corn oil was used. Considerable amounts of free amino acids were obtained after the biodegradation of feather which makes the new strains promising for application in feather-waste treatment to, e.g., transformation to animal feedstuff.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Complementation between uvr mutants of Streptococcus pyogenes

Summary From the group A streptococcal strains K 56 and 56 188, respectively 13 and 6 nitrosoguanidine-induced uvr mutants were isolated and used in complementation experiments employing strain 56 188 and its derivatives as donors in phage A 25-mediated heterologous transductions. When A 25 propagated on wild type or on 2 of the six 56 188-derived uvr mutants was used to infect 4 of the uvr recipient strains, a substantial increase in survivors of UV irradiation was found over those observed in selfing experiments or control experiments without phage. Less than 1% of the UV survivors had stably integrated the uvr ⁺ allele. The remaining 4 uvr donors failed to complement the 4 above-mentioned recipients, indicating that the strains in question fell into 2 complementation groups.Nine of the 13 K 56-derived mutants, which in contrast to the others were characterized by non-reversibility to UV resistance, did not even show an increase in UV survivors when infected with phage grown on wild type. The possibility is discussed that these strains might carry second mutations affecting UV sensitivity which, however, did not appear to be of the rec type.     

Detection of<Emphasis Type="Italic">pap</Emphasis>-,<Emphasis Type="Italic">sfa</Emphasis>- and<Emphasis Type="Italic">afa</Emphasis>-specific DNA sequences in<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from extraintestinal material

P-fimbriae, S-fimbriae and AFA-adhesins are virulence factors responsible for adherence ofEscherichia coli strains to extraintestinal host-cell surface. Detection ofpap-,sfa- andafa-specific sequences performed by PCR revealed 74%pap ⁺, 65%sfa ⁺, and 8.3%afa ⁺ strains in a group of 84 extraintestialE. coli isolates. Detection in a group of fecal strains showed 29%pap ⁺, 21%sfa ⁺ and 4%afa ⁺ strains.pap together withsfa were found as the most frequent combination (56%) among extraintestinal isolates probably due to localization ofpap-andsfa-operons on a common pathogenicity island. The occurrence ofafa-specific sequence among 56 urine strains was 11%, although noafa ⁺ strain was detected among 28 gynecological isolates. No strains with detected adhesin operons were found among twenty (24%) extraintestinalE. coli strains.     

Cloning,expression and characterization of attachment‐invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays

Aims: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment‐invasion locus (Ail) protein. Methods and Results: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l^?1 final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. Conclusion: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. Significance and Impact of the Study: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.