响应面法优化波赛链霉菌JMC 06001发酵培养基

采用响应面法对产生抑菌活性物质的波赛链霉菌（Streptomyces peucetius）菌株JMC 06001的发酵培养基进行优化。首先采用Minnimum Run Equireplicated Res IV设计对初始发酵培养基的8个营养因素进行筛选,获得影响产生抑菌活性物质的3个主要影响因素：葡萄糖、大豆粉和NaCI;然后用最陡爬坡实验快速逼近最大响应区域;最后,结合Box-Behnken设计及响应面分析,确定主要影响因素的最佳浓度,得出该菌株产抑菌活性物质的最优发酵培养基配方为：葡萄糖1．2％,麦芽糖0．7％,蛋白胨0．9％,大豆粉1．4％,NaCl3．7％,CaCO3 0．1％,复合盐A液2．0％,复合盐B液0．1％,起始pH值7．0。用优化后的培养基发酵所得发酵液对敏感指示菌藤黄八叠球菌的抑菌圈直径达31．5mm,与预测值的相对偏差仅为1．59％,与用初始发酵培养基发酵所得发酵液的抑菌效果（抑菌圈直径26．5mm）相比提高了18．9％。     

波赛链霉菌JMC 06001抑菌活性物质的特性研究

使用藤黄八叠球菌（Sarcina lutea）、枯草芽胞杆菌（Bacillus subtilis）、大肠埃希菌（Escherichia coli）、黑曲霉（Asperillus niger）、产气肠杆菌（Enterobacter aerogenes）、变形杆菌（Proteus vulgaris）、白色念珠菌（Candidaalbicans）、青霉（Penicilliumsp.）、铜绿假单胞菌（P.aeruginosa）9种指示菌,采用杯碟法检测抑菌活性。研究了波赛链霉菌JMC 06001菌株（Streptomyces peucetiusJMC 06001）发酵液和菌丝体中活性物质的抑菌谱,对由该菌株产生的抑菌物质的温度、pH值、抗紫外线方面的稳定性进行了考察。利用硅胶柱层析和薄层层析对抑菌物质的分离也做了初步的研究。结果表明,该菌株的发酵产物对部分革兰阳性菌、革兰阴性菌和部分真菌有较为明显的抑制作用。稳定性试验结果显示,该菌株产生抑菌活性物质的最适温度为40℃左右,最佳pH值在7.0左右,具有较好的热稳定性和较宽的pH作用范围,抗紫外线辐射能力较强。通过硅胶柱层析分离和薄层色谱检测,显示该菌次生代谢产物中的抑菌活性物质包含了多种成分。     

The DrrC protein of Streptomyces peucetius, a UvrA-like protein, is a DNA-binding protein whose gene is induced by daunorubicin

DrrC, a daunorubicin resistance protein with a strong sequence similarity to the UvrA protein involved in excision repair of DNA, is induced by daunorubicin in Streptomyces peucetius and behaves like an ATP-dependent, DNA binding protein in vitro. The refolded protein obtained from expression of the drrC gene in Escherichia coli was used to conduct gel retardation assays. DrrC bound a DNA segment containing the promoter region of a daunorubicin production gene only in the presence of ATP and daunorubicin. This result suggests that DrrC is a novel type of drug self-resistance protein with DNA binding properties like those of UvrA. Western blotting analysis with a polyclonal antiserum generated against His-tagged DrrC showed that the appearance of DrrC in S. peucetius is coincident with the onset of daunorubicin production and that the drrC gene is induced by daunorubicin. These data also showed that the DnrN and DnrI regulatory proteins are required for drrC expression. The level of DrrA, another daunorubicin resistance protein that resembles ATP-dependent bacterial antiporters, was regulated in the same way as that of DrrC.     

Genetic engineering of doxorubicin production in Streptomyces peucetius: a review

The genetics and biochemistry of daunorubicin and doxorubicin production by Streptomyces peucetius is reviewed, with a focus on how such information can be used for the genetic engineering of strains having improved titers of these two antitumor antibiotics. Received 23 February 1999/ Accepted in revised form 22 March 1999     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究

通过对14株蛹拟青霉菌株进行摇瓶液体发酵培养试验,筛选虫草素产量最高的蛹虫草菌株,并确定了虫草素的最佳收获期;比较8种营养添加物对虫草发酵过程中虫草素积累的影响,筛选出最佳的营养添加物,以提高液体发酵生产虫草素的产量。结果表明：蛹虫草CM001号菌株的虫草素产量最高,蛹虫草菌在第10天细胞量达到最大值20.44mg/mL,虫草素含量在第13天达到最大值73.81mg/L,70%以上的虫草素分布在发酵液中。其中分别添加腺苷、腺嘌呤、丙氨酸、L-天冬氨酸、甘氨酸5种物质,对虫草素合成的促进作用均较明显,均能有效提高蛹虫草液体发酵虫草素的产量,特别是添加1g/L的腺嘌呤能使10号菌株的虫草素产量提高7.09倍。     

Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66

A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin 3 (rHuIL-3) by Streptomyces lividans. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of 20 to 8. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation. Following this procedure, the search was narrowed to four amino acids (Asp, Leu, Met, and Phe). Finally, a mixture design experiment known as the simplex lattice design was carried out and the composition of the optimum minimal medium was found (Asp 53%, Met 5%, and Phe 42%).     

Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans

A thermophilic strain of Streptomyces thermonitrificans produced a high activity of intracellular glucose isomerase (12 U/ml) when grown in a medium containing 1% (w/v) xylose, supplemented with 2% (w/v) sorbitol as the second carbon source, at 50°C for 16 h. Addition of Mg²⁺ enhanced enzyme production but the organism could grow and produce the enzyme in the absence of Co²⁺.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, IndiaNCL Communication No. 5813     

High-yield production of lipoglycopeptide antibiotic A40926 using a mutant strain Nonomuraea sp. DP-13 in optimized medium

The lipoglycopeptide antibiotic A40926 produced by Nonomuraea sp. is a complex of structurally related components differing in the fatty acid moiety. Besides showing an intrinsic antibacterial activity, A40926 is the precursor of the semisynthetic antibiotic Dalvance. In this work, A40926 production by a mutant strain Nonomuraea sp. DP-13 was investigated. It was found that A40926 production was markedly promoted by using poorly assimilated carbon source maltodextrin and nitrogen source soybean meal. Addition of Cu²⁺ resulted in a stimulation of A40926 production, but Co²⁺ had an inhibitory effect. L-Leucine addition greatly improved total A40926 production and modified the complex composition toward factor B0. An optimized production medium IM-3 was developed and a maximum A40926 production of 1096 mg/L was obtained in the 10-L fermenter. This was the highest A40926 productivity so far reported.     

Optimisation of medium composition for clavulanic acid production by Streptomyces clavuligerus

Among four different commercially available nitrogen sources containing soybean derivatives, a protein extract of soybean gave the highest yield for clavulanic acid production by Streptomyces clavuligerus. A statistical method based on factorial design of experiments was applied to optimise the medium. An empirical model was obtained by applying response surface statistical analysis. The analysis of variance showed that concentrations of protein extract of soybean and glycerol and the interaction between these two variables were significant at 95% level of confidence. The maximum clavulanic acid concentration obtained in 72 h was 1.2 g l^–1.     

Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnmT genes involved in doxorubicin (adriamycin) biosynthesis.

Sequence analysis of a 3.4-kb region Streptomyces peucetius daunorubicin (DNR) gene cluster established the presence of the dnrH and dnmT genes. In dnrH mutants, DNR production increased 8.5-fold, compared with that in the wild-type strain, while dnmT mutants accumulated epsilon-rhodomycinone (RHO), which normally becomes glycosylated in daunorubicin biosynthesis. Hence, dnmT may be involved in the biosynthesis or attachment of daunosamine to RHO or in the regulation of this process. Since the DnrH protein is similar to known glycosyl transferases, this protein may catalyze the conversion of DNR to its polyglycosylated forms, known as baumycins. Overexpression of dnmT in the wild-type and dnrH mutant strains resulted in a major decrease in RHO accumulation and increase in DNR production.     

Enhanced production of avermectin B1a by medium optimization and glucose feeding with Streptomyces avermilitis

Avermectin B1a is an important macrolide antibiotic with potent anthlemintic and insecticidal activity. The objective of this study was to enhance productivity of avermectin B1a by optimizing culture medium and glucose feeding fermentation. Results showed that B1a concentration was increased by 48.6% through optimization of nitrogen, carbon sources as well as supplement of 0.2 mM Co²⁺. It was found that a maximum B1a concentration of 826 mg/l was attained by glucose-feeding in a laboratory scale fermentor, which was two-fold higher than that in the control fermentation. Meanwhile, the proportion of B1a in the total products was increased by 6% with respect to the control process. These results would be very useful for maximizing productivity of avermectin B1a in an up-scaled fermentation.     

Studies on the production of actinomycin-D by Streptomyces griseoruber– a novel source

Aims:  To isolate and characterize bioactive metabolites produced by a micro-organism isolated from a soil sample associated with the roots of a medicinal plant, Azadirachta indica .
Methods and Results:  Morphological, cultural, physiological and 16S rRNA homology studies revealed that the organism showed 99% similarity with Streptomyces griseoruber NBRC 12873. One bioactive metabolite (Py2) isolated from the fermented broth was characterized as actinomycin-D (act-D). It showed high activity against various Gram-positive and Gram-negative bacterial cultures, Mycobacterium tuberculosis H37Rv and human neoplastic cells in vitro using standard protocols.
Conclusions:  The isolated strain S. griseoruber produced act-D predominantly (210 mg l⁻¹, c. 88% of the crude) under nonoptimized growth conditions.
Significance and Impact of the Study:  Streptomyces griseoruber may be exploited as a potential source for the commercial production of act-D, as this strain is not reported to produce act-D. Further investigations on the strain for commercial application will be of immense pharmaceutical importance.     

Isolation,taxonomic and fermentation studies on a new strain of Streptomyces arenae var ukrainiana producing a tetraene antibiotic

A Streptomyces strain UK10 was isolated from Ukrainian soil and identified by taxonomical studies as Streptomyces arenae var ukrainiana. HA-2-91 was isolated from the biomass of S. arenae var ukrainiana and is supposedly a polyene macrolide antibiotic belonging to the tetraene group. HA-2-91 showed promising antifungal activity (in vitro) against yeasts and filamentous fungi, including plant pathogens and dermatophytes and was found to be less toxic in mice than nystatin and rimocidin.     

Improvement of pristinamycin production by genome shuffling and medium optimization for Streptomyces pristinaespiralis

To isolate an improved pristinamycin producing strain of Streptomyces pristinaespiralis, the technique of Genome shuffling was used which resulted in a high-yield recombinant G 3-56 strain. Strain G 3-56 yielded 322 ± 17 mg/L of pristinamycin which was 11.4-fold higher than that of the initial strain and 3.7-fold higher than strain UN-78 which previously had the highest yield of pristinamycin. The genetic characteristics of the recombinant G 3-56 strain was stable as revealed by our subculture experiments. The optimal production medium was determined using the orthogonal matrix method. Under the optimal medium conditions, the maximum yield of pristinamycin was 412 mg/L with about 1.24-fold higher than the original medium.     

常压室温等离子体(ARTP)诱变及高通量筛选那西肽高产菌株

采用新型常压室温等离子体(ARTP)诱变活跃链霉菌(Streptomyces actuosu),并应用抑菌圈和48孔板培养方法高通量筛选高产那西肽菌株。研究表明抑菌圈径的大小与48孔板效价之间以及48孔板效价与摇瓶效价之间均有较好的相关性,系数R分别达到0.534和0.896。通过多轮ARTP诱变及高通量筛选最终获得了3株相对效价提高50%以上的遗传性能稳定的突变株。ARTP诱变技术作为获得那西肽高产菌株的有效途径,与传统摇瓶发酵筛选相比,48孔板及抑菌圈法能显著提高那西肽高产菌株的筛选效率。     

高产精氨酸菌株选育及发酵培养基优化

利用改良阿须贝培养基筛选出4种产精氨酸的菌株,其菌落特征分别为白色链球状菌株,干白分散球状菌株,金黄色杆状菌株,粉红色球状菌株,对它们分别进行了产精氨酸发酵试验,利用分光光度计测定精氨酸含量,最终确定金黄色棒状菌株为生产精氨酸产量较高的菌株。通过单因素和正交设计试验,分别考察玉米浆、硫酸铵、尿素、葡萄糖对金黄色杆状菌株发酵产精氨酸的影响,结果表明最佳条件为:玉米浆浓度3%,硫酸铵浓度6%,尿素浓度0.3%,葡萄糖浓度13%,在此条件下,可获得精氨酸最大产量为368.9ug/mL,与初始发酵培养基条件下发酵相比精氨酸产量提高约300%。该研究为精氨酸菌株的筛选提供了一条新途径,也为所筛选的金黄色杆状菌后期的研究及进一步规模生产精氨酸提供了参考依据。