Increase in pladienolide D production rate using a Streptomyces strain overexpressing a cytochrome P450 gene

Pladienolide B and its 16-hydroxylated derivative (pladienolide D) are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107 showing strong in vitro and in vivo antitumor activity. While pladienolide B is mainly produced by this strain, pladienolide D is produced to a lesser extent. To facilitate the production of pladienolide D by biotransformation, we found that Streptomyces bungoensis A-1544 was able to hydroxylate pladienolide B at 16-position. We identified psmA from S. bungoensis A-1544, which encoded a pladienolide B 16-hydroxylase PsmA belonging to the CYP105 family of cytochrome P450. To increase the efficiency of pladienolide D production, we constructed recombinant S. bungoensis A-1544 overexpressing psmA and performed biotransformation of pladienolide B to pladienolide D. This biotransformation achieved a production level 15-fold higher than that using the control strain S. bungoensis A-1544/pIJ702.     

Involvement of cytochrome P450 in hydroxylation of propylbenzene by Fusarium moniliforme strain MS31

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.     

Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis

The global regulatory gene, afsR2, from Streptomyces lividans was previously reported to highly stimulate two structurally unrelated antibiotics, actinorhodin and undecylprodigiosin, in both S. lividans and its close relative S. coelicolor. Production of eight avermectin components was also improved in S. avermitilis: the use of wild-type S. avermitilis and its high-producing mutant, transformed by introduction of multiple copies of afsR2, increased the total avermectin productions by 2.3-fold and 1.5-fold, respectively.     

Anthocyanins and their metabolites are weak inhibitors of cytochrome P450 3A4

The cytochrome P450 enzyme cytochrome P450 3A4 (CYP3A4) controls the metabolism of about 60% of all drugs, and its inhibition may dramatically affect drug safety. Modulation of cytochrome P450 activity has been observed by constituents of fruit extracts including several flavonoids. The present investigation addresses CYP3A4 inhibition by anthocyanins, their aglycons, proanthocyanidins, and phenolic metabolites using a chemiluminescent assay. Test compounds inhibited CYP3A4 activity in a concentration-dependent manner featuring IC(50) values from 12.2 up to 7,842 microM. In the order of decreasing effect size, anthocyanidins were followed by anthocyanins, proanthocyanidins, and phenolic acids. When compared to earlier data on furanocoumarins from grapefruit extract, the inhibitory activity of tested anthocyanins, and anthocyanidins was shown to be about 10,000-fold weaker, and was negligible for phenolic acids (>100 000-fold weaker). Future studies are invited to address effects of the above flavonoids on other CYP isoforms for more detailed toxicity profiles.     

Behaviour ofYersinia enterocolitica andAeromonas hydrophila in skim milk during fermentation by various lactobacilli

In this study, behaviour ofYersinia enterocolitica andAeromonas hydrophila in skim milk during fermentation by various_{lactobacillus} sp. were determined. pH values of the skim milk samples were also examined during fermentation. The amount of produced lactic acid and diacetyl/acetoin productions of theLactobacillus sp. were estimated. Antimicrobial effects of the lactobacilli onY. enterocolitica andA. hydrophila were also determined by an agar diffusion method. WhileY. enterocolitica was not inhibited and grew during fermentation,A. hydrophila was inhibited, in part, and the growth was retarded. Results were supported by the agar diffusion method forY. enterocolitica, whereas inhibition activity was not found forA. hydrophila. The highest lactic acid productions were estimated inL. bulgaricus (7.50 mg/ml) andL. acidophilus (5.63 mg/ml) and four out of sixLactobacillus sp. were found to be diacetyl/acetoin producers.     

Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471

Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50–55 °C, and the enzyme was stable from pH 5 to 10 and up to 37 °C. The SMC activity requires Mg²⁺ or Mn²⁺ and the order of potency to enhance the activity was Zn²⁺ ≥ Mn²⁺ > Cu²⁺ ≥ Fe²⁺. Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H₂N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.     

Fermentation of rice using amylolytic Bifidobacterium

Twenty-two amylolytic bifidobacteria grown in BHI-starch media were compared for the amylase activity of the intra- and extra-cellular enzymes. The activity of the cells grown in the liquid medium differed considerably. Among the strains tested B. adolescentis Int57 and B. adolescentis ZS8 exhibited higher activities than others. In rice medium containing 0.05% -cysteine·HCl and 0.2% yeast extract, the amylolytic Bifidobacterium strains grew considerably better than non-amylolytic Bifidobacterium strains. B. adolescentis Int57, which showed highest growth and amylase activity in the rice medium, was chosen and rice fermentation was investigated. The titratable acidity (TA) reached 2.43 and pH decreased to 4.4 after 24 h fermentation. The relative ratio of acetic acid to lactic acid gradually decreased during fermentation. The concentration of reducing sugar and amylase activity gradually increased and reached 14 mg maltose equivalent/ml and 35 mU/ml min, respectively, in 24 h. The accumulated reducing sugar was mostly maltotriose. The layer-separation of fermented product was stabilized by the addition of 1% gelatin. It was suggested that amylolytic bifidobacteria may be used for the production of fermented rice products.     

Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica

A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60·2 kDa in molecular mass, and had 30–45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450Alk to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica. © 1998 John Wiley & Sons, Ltd.     

Bioconversion of the antihistaminc drug loratadine by tobacco cell suspension cultures expressing human cytochrome P450 3A4

In this study we have expanded the metabolic potential of plant cell suspension cultures by introducing active human cytochrome P450 monooxygenase 3A4 into tobacco cells. Exogenously supplied loratadine was metabolized in a 3A4-specific manner, showing the capacity of this system for the generation of metabolites.     

植物乳杆菌发酵桑葚酵素工艺优化及质量评价

目的:解决新鲜桑葚难以保存的问题,将新鲜桑葚制成桑葚酵素。方法:以新鲜桑葚汁为原料,植物乳杆菌为生产菌种,总酚含量为评价指标,通过单因素试验结合响应面试验优化了植物乳杆菌桑葚酵素的发酵工艺,并对桑葚酵素的理化、微生物及感官质量指标进行评价。结果:优化后发酵工艺条件为发酵时间40 h、发酵温度32℃、接种量25%,所制得的植物乳杆菌桑葚酵素的总酚含量达(43.48±0.67)μg/mL,是未经发酵的桑葚汁的1.62倍,可溶性固体物含量为5.36%,pH值为4.08±0.01,微生物指标满足国家标准;桑葚酵素呈紫红、色泽均匀,具有浓郁的桑葚果香和发酵的香味、无异味,酸味柔和、风味好,有光泽、无杂质及沉淀。结论:经植物乳杆菌发酵制得桑葚酵素的过程有生物活性物质产生,有利于提高桑葚酵素质量。     

Identification of novel cytochrome P450 monooxygenases from actinomycetes capable of intermolecular oxidative C–C coupling reactions

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Immunization of the industrial fermentation starter culture strain of Saccharomyces cerevisiae to a contaminating killer toxin-producing Candida tropicalis

K3 killer trait was introduced into the fermentation starter strain of Saccharomyces cerevisiae BSP 1 in order to construct immune industrial strain that produces K3 type killer toxin and was resistant to Candida tropicalis (K⁺) contamination. Protoplasts of respiration-deficient Rho^o strain of S. cerevisiae NCYC 761 (K3) and S. cerevisiae BSP 1 were fused. The resulting respiration-competent hybrid with K3 type killer activity was selected on media containing a non-fermentable carbon source and by a killer zone assay in a plate test, respectively. The fusant was similar to the parent strain in its fermentation and sugar utilization patterns, growth rate, dough-raising properties and osmotolerance. The newly constructed S. cerevisiae BSP 1 (K3) inhibited the growth of C. tropicalis in a pH range from 3.5 to 5.0 and over a temperature range of 20–30°C.     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

产γ-亚麻酸诱变菌拉曼被孢霉HLY0902的发酵条件优化

采用正交实验和单因素实验,分别对诱变菌拉曼被孢霉HLY0902的培养基及培养条件进行优化,以期提高γ-亚麻酸(GLA)的产量。结果表明:当发酵培养基组成为葡萄糖100 g/L、酵母浸粉10 g/L、KH_2PO_44 g/L、Na NO_31 g/L、Mg SO_4·7H_2O 0.5 g/L时,GLA的产量最大,可达1.05g/L,较优化前提高了43.8%;最优培养条件为接种量10%,装液量20%,发酵时间168 h,适合菌体生长和油脂积累的p H为5.5、发酵温度为22℃,适合GLA积累的p H为7.5、发酵温度为20℃。通过该系列的优化研究,诱变菌拉曼被孢霉HLY0902产GLA的能力显著提高。     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of protein hydrolysates from Phaseolus lunatus and Phaseolus vulgaris seeds

Phaseolus lunatus and Phaseolus vulgaris protein concentrates were hydrolyzed with the enzymes Alcalase^® and Flavourzyme^® at different reaction times, and the angiotensin-I converting enzyme (ACE-I) inhibitory activity, antioxidant properties and amino acid composition measured in the hydrolysates. With Alcalase^®, the highest degree of hydrolysis (DH) in P. lunatus was 37.94% at 45 min, and in P. vulgaris was 49.48% at 30 min. With Flavourzyme^®, the highest DH's were 22.03% and 26.05%, respectively, both at 90 min. ACE-I inhibitory activity in the Alcalase^® hydrolysates was IC₅₀ = 0.056 mg mL⁻¹ for P. lunatus at 90 min, and IC₅₀ = 0.061 mg mL⁻¹ for P. vulgaris at 60 min. In the Flavourzyme^® hydrolysates this activity was IC₅₀ = 0.0069 mg mL⁻¹ for P. lunatus at 90 min and IC₅₀ = 0.127 mg mL⁻¹ for P. vulgaris at 45 min. In SDS-PAGE, the hydrolysates exhibited low molecular weight bands. Antioxidant activity was 11.55 mmol L⁻¹ TEAC mg⁻¹ protein for P. lunatus with Flavourzyme^® at 90 min and 10.09 mmol L⁻¹ TEAC mg⁻¹ protein for P. vulgaris with Alcalase^® at 60 min. Amino acid composition exhibited high amino acid hydrophobic residues content.     

鸡源大肠埃希氏菌mcr-1基因携带及分析

目的 了解在农业农村部禁止使用多黏菌素作为动物促生长使用后四川部分地区鸡源大肠埃希氏菌（E.coli）mcr-1基因的携带情况,为制定进一步防控措施提供依据。方法 采集四川部分地区市场售卖点肉鸡直肠拭子,用含有多黏菌素（终浓度4 μg/mL）的EC肉汤增菌接种含多黏菌素（终浓度4 μg/mL）的麦康凯平板,挑取可疑菌落,采用PCR方法鉴定菌株并检测mcr-1基因;微量肉汤稀释法测定mcr-1基因阳性菌株对临床常见抗菌药物耐药情况。脉冲场凝胶电泳（PFGE）对mcr-1基因阳性菌株进行同源分析。耐药基因质粒结合实验验证mcr-1基因传播途径。结果 从70份肉鸡样本中的13份检出mcr-1基因阳性大肠埃希氏菌,检出率18.57%（13/70）,对实验的13种抗生素,除13株mcr-1阳性菌株对头孢西丁有12株敏感以外,对其他抗生素都表现出不同程度的耐药,其中四环素和甲氧苄啶/磺胺甲恶唑耐药率最高,达到了100%（13/13）;其次是氨苄西林和氯霉素,耐药率为84.62%（11/13）。PFGE显示13株mcr-1阳性大肠埃希氏菌分属13个不同的型别;质粒结合实验显示mcr-1基因能够通过质粒传播。结论 mcr-1基因在鸡大肠内大肠杆菌中检测率比较高,且鸡大肠中mcr-1阳性大肠埃希氏菌的耐药情况比较严重。     

Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis

NADPH cytochrome P450 oxidoreductase (CPR) catalyses the transfer of electrons during P450-mediated oxidation, which plays an important role in the omega-oxidation pathway of Candida tropicalis. Two putative allelic genes, CPR-a and CPR-b, were cloned from the long chain dicarboxylic acid-producing Candida tropicalis 1230, using cassette PCR methods. Both the identified open reading frames predict the gene products of 679 amino acid residues. The deduced amino acid sequences of CPR-a and CPR-b are highly homologous to CPR genes from C. tropicalis ATCC 750 and Candida maltosa. Both genes were individually expressed in a cpr mutant of Saccharomyces cerevisiae with high CPR activities, in which only a small distinction was observed between recombinant CPR-a and CPR-b. Both CPR-a and CPR-b contain one CTG codon, which codes for serine (amino acid 50) in C. tropicalis rather than universal leucine. A mutated cDNA of CPR-a with a TCG codon instead of CTG codon was constructed and expressed, resulting in little increase in CPR activity. This indicates that the alteration of Ser-50 has little effect on functional expression of CPR. Furthermore, high ketoconazole sensitivity for the cpr mutant was complemented by heterologous expression of the cloned CPR-a or CPR-b.