Lovastatin biosynthesis by Aspergillus terreus in a chemically defined medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Regulation of neuroblast proliferation by hormones and growth factors in chemically defined medium

Pure neuronal cultures prepared from 6-day-old embryonic chick brains incorporated [3H]-thymidine in serum-free medium up to the 4th day in culture. The addition of insulin any time within this culture period caused an increase in thymidine incorporation. This increase in [3H]-thymidine was correlated with an increase in cell number and percentage of labeling index. Triiodothyronine and endothelial cell growth factor were also active in stimulating [3H]-thymidine incorporation into chick neuroblasts. The effect of these trophic agents is unique since a variety of known mitogens tested were negative.     

Cultivation of Pasteurella haemolytica in a chemically defined medium

A chemically defined medium was developed for the aerobic cultivation of Pasteurella haemolytica. Studies on the growth of strain H44L were conducted in a medium consisting of 15 amino acids, inorganic salts, citrate, nicotinamide, pantothenate, thiamine or thiamine monophosphate, and carbon sources. The amino acids were provided as l isomers, because racemic mixtures of some amino acids inhibited growth. The carbon source consisted of a mixture of 1.0% d-galactose and 0.1% d-glucose. Culture populations of strain H44L reached 2 x 10(10) cells per milliliter after 16 hr of incubation at 37.5 C. Other strains of P. haemolytica, from a wide variety of sources, were tested for growth in the medium, and 23 of 24 strains grew well. Five strains of P. haemolytica var. ureae failed to grow in the medium.     

发酵法生产丁二酸的化学合成培养基的筛选

以产琥珀酸放线杆菌Actinobacillus succinogenes NJ113 为出发菌株,针对该菌株筛选出含有关键生长因子的化学合成培养基,其关键因子为谷氨酸（Glu）、蛋氨酸（Met）和生物素（VH）和烟酸（VPP）。结合原发酵培养基中的磷酸缓冲盐成分,最终得到的化学合成培养基配方（g/L）： CH3COONa 1.36,NaCl 1.0,MgCl2 0.2,CaCl2 0.2,Na2HPO4 0.31,NaH2PO4 1.6, KH2PO4 3,NH4HCO3 1.57,Glu 0.87,Met 0.11,VH 0.010,VPP 0.025。在3 L发酵罐上进行验证实验,50 g/L初始葡萄糖发酵70 h,丁二酸的质量浓度为45.2 g/L,丁二酸收率达到90.4%。与之前的半合成培养基发酵制备丁二酸相比,丁二酸的收率提高了25.2%,副产物也有很大幅度的减少。     

Nutritional studies on Acanthamoeba culbertsoni and development of chemically defined medium

A chemically defined medium containing 11 amino acids, 3 vitamins, 6 inorganic salts and glucose, yielding maximum cell densities of 1.5-2.5 x 10(7) cells/ml, has been developed for Acanthamoeba culbertsoni with a mean generation time (MGT) of 10 h. A medium containing six amino acids viz. arginine, methionine, leucine, isoleucine, valine and glycine along with other components could also support good albeit slower growth (MGT 27 h) of the amoeba. Acetate did not serve as a suitable carbon/energy source for A. culbertsoni. This organism bears close resemblance in its nutritional requirements to other Acanthamoeba especially A. polyphaga.     

A chemically defined and minimal medium for Clostridium difficile

A chemically defined medium (CDCDM) has been developed for Clostridium difficile. The medium contains nine amino acids, five mineral salts, N -acetylglucosamine and the growth factors riboflavin and nicotinic acid. Ten strains of C. difficile have been subcultured repeatedly in this medium with no apparent changes in colonial or cellular morphology. The metabolic end-products of strains grown in this medium were reproducible and yielded patterns similar to those produced by cells cultured in Brain Heart Infusion broth (BHI). The growth rates were approximately 40% slower than those in a complex medium and the growth rate constants ranged between 0·011 and 0·087 h^-1. When the defined medium was supplemented with proteose peptone, yeast extract or caesin hydrolysate at concentrations of 1%, growth increased. No such growth increase was observed when the medium was supplemented with casamino acids or glucose.     

Embryonic stem cell development in a chemically defined medium

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.     

Effect of precursors on biosynthesis of monensins A and B

Precursors of monensins (acetate, propionate, butyrate, isobutyrate) affect the total production and the relative proportion of monensins A and B. Addition of propionate into the fermentation medium causes a prevalence of monensin B whereas butyrate and isobutyrate stimulate the production of monensin A and suppress the production of monensin B.     

Antibacterial testing of metal ions using a chemically defined medium

Data from in-vitro tests on potential germicides can be greatly influenced by the culture medium. The bioavailability and biochemical reactivity of the biocides can be influenced by chemical interference with media components (Spooner & Sykes 1972). Bird et al . (1985) showed that metal ions are particularly prone to chemical interferences. A chemically defined solid medium has been developed to monitor the antibacterial activity of metal ions. The minimum inhibitory concentrations of zinc and silver have been determined against a range of bacteria using this medium.     

Development of a chemically defined medium for growth ofNeisseria gonorrhoeae

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO₂ during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.This investigation was supported by a Public Health Service Predoctoral Fellowship (F-FI-GM-24-755-01A1) from the National Institute of General Medical Sciences of the United States Public Health Service to the senior author.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.     

Synthesis of sterigmatocystin on a chemically defined medium by species ofAspergillus andChaetomium

Sterigmatocystin (ST) is a secondary metabolite and a principal mycotoxin known to be produced by over 30 species of filamentous fungi. It is also one of the late intermediates in aflatoxin biosynthesis. We have tested the ability of 7 species ofAspergillus, including 4 strains ofA. versicolor, one species ofBipolaris, and two species ofChaetomium, to produce ST on a sucrose-salts-phenylalanine defined medium as well as on three complex substrates. Highest ST production in our survey was by a strain ofA. versicolor grown on wheat, whereas, the highest ST production on defined medium was byC. cellulolyticum. To our knowledge, this is the first report of ST production byC. cellulolyticum on any substrate. In precursor feeding studies, resting cultures of wild typeA. nidulans andA. versicolor were unable to biotransform O-methylsterigmatocystin (OMST), the last known intermediate in aflatoxin biosynthesis. These results suggest that ST is the end product of polyketide metabolism in the strains tested.     

Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium

A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively. The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid. In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes. The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum. Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes. In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability. Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures. The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media. The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay. Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium. However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.     

A chemically defined fermentation medium for the growth of Micromonospora purpurea

A chemically defined medium for Micromonospora purpurea has been devised, consisting of glucose, a nitrogen source, calcium carbonate, magnesium sulfate, dibasic potassium phosphate, and the required trace quantities of iron, copper, zinc, and manganese. Using washed cell inocula, dry mycelial weights of more than 16 mg./ml. were obtained in 7-day shaken-flask fermentations. Nutrient requirements for M. purpurea are discussed and growth data presented. Sucrose, maltose, starches and dextrins could be substituted for glucose, and resulted in good growth of the organism. A number of amino acids and inorganic nitrogen-containing compounds were capable of utilization as sole nitrogen sources. Weekly serial transfers of the culture in defined medium have shown no diminution in mycelial weights over a four-month period.