Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213

In beta-lactam producing microorganisms, the first step in the biosynthesis of the beta-lactam ring is the condensation of three amino acid precursors: alpha-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, alpha-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.     

Enhancement of cephamycin C production using soybean oil as the sole carbon source

Vegetable oils were investigated to evaluate their potential to act as the sole carbon source for production of cephamycin C in shake and jar-fermentor cultures. Soybean oil was the best carbon source for cephamycin C production. Bioautography and HPLC analyses showed that cephamycin C was exclusively produced even when soybean oil was used as the sole cabon source. The optimal pH and initial concentration of soybean oil was 7.5 and 7 g/l, respectively. Both pH and the pH-control agent affected cephamycin C production, and among phosphoric acid, acetic acid and sulfuric acid, phosphoric acid was associated with the best production. Soybean oil was slowly consumed after the soluble nitrogen source was consumed. When the initial soybean oil concentration was 7 g/l, cephamycin C production was maximal, 2.0 g/l, which was twice as high as that from starch. The product yield from soybean oil was 4.7 times higher than that from starch. These results show that vegetable oils, which are cheaper than other carbon sources, could be used as the sole carbon source in the production of antibiotics. Correspondence to: M. Okabe     

Cephamycin C biosynthesis in Streptomyces cattleya: nitrogen source regulation

Summary The production of cephamycin C by Streptomyces cattleya varies with the use of asparagine, glutamine or ammonium as nitrogen sources. hydroxylase and expandase activities were demonstrated for the first time with this species. A study of the biosynthetic regulation of these enzymes by two different nitrogen sources, glutamine and asparagine, was carried out. Asparagine proved to be a better nitrogen source, both for enzymatic biosynthesis and production of cephamycin C. Moreover, an excess of asparagine in the culture environment provokes, simultaneously, a reduction in cephamycin C production and a decrease in the biosynthesis of expandase and hydroxylase.Offprint requests to: A. Lebrihi     

Nutritional requirements ofLysobacter lactamgenus for the production of cephabacins

Summary To optimize the fermentation medium for the production of new cephem compounds, cephabacins, by an eubacteriaLysobacter lactamgenus IFO 14,288, the effects of medium components on cephabacin production were investigated. Supplementation of glucose as a sole carbon source in liquid media was the best for the antibiotic production as well as for the cell growth. Casamino acid was the best nitrogen source for antibiotic biosynthesis and cell growth among nitrogen sources tested, and this strain could utilize sulfate or thiosulfate as a sulfur source. No significant effects of growth factors (vitamins) on the antibiotic production and cell growth were observed, but ferrous, magnesium and nickel ions slightly enhanced the cephabacin production.     

Effect of nutritional factors on lipase biosynthesis by Aspergillus niger

Summary Lipase biosynthesis occured in medium without lipids, but for improved production an inducer was needed. The source and concentration of an inducer had no signifficant effect. Starch as an additional carbon source stimulated lipase biosynthesis when used in small amounts. Addition of NH₄NO₃ as a nitrogen source, KH₂PO₄ as a phosphate source as well as Mg ions to the medium with inital pH 5.0 gave the best yield.     

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.     

Dissolved oxygen control of a maltose feed to batch fermentations ofStreptomyces clavuligerus: Effect on cephamycin C biosynthesis

Summary Compared to controls, a maltose-fed fermentation ofStreptomyces clavuligerus showed a 2-fold reduction in desacetoxycephalosporin C synthase activity and in the production of the antibiotic, cephamycin C. Accumulation of the pathway intermediate, penicillin N occurred in the control fermentations but not in the maltose-fed culture, indicating that the carbon source was also regulating steps earlier in the pathway.Since the dissolved oxygen concentration was effectively maintained at almost constant levels in both the controls and maltose-fed fermentations, the observed maltose interference with cephamycin C biosynthesis was not related to the aeration condition of the actively growingS. clavuligerus culture.     

Novel electrochemical-enzymatic model which quantifies the effect of the solution Eh on the kinetics of ferrous iron oxidation with Acidithiobacillus ferrooxidans

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.     

溶剂稳定性蛋白酶产生菌Bacillus licheniformis YP1的发酵优化

溶剂稳定性蛋白酶产生菌Bacillus licheniformis YP1分离自油田土样。考察了碳源、氮源、金属离子等营养因素对YP1菌株发酵产溶剂稳定性蛋白酶的影响。YP1菌株发酵产胞外蛋白酶的最佳碳源为淀粉,果糖、甘露糖和乳糖显著抑制产酶;最佳氮源为酵母膏,干酪素、酵母粉和牛肉膏促进产酶,玉米浆和尿素显著抑制产酶。Mn^2＋可以显著促进酶活,Mg^2＋可以促进产酶,在初步优化的培养条件下,YP1菌株的胞外蛋白酶产量达980U。     

甲壳素脱乙酰酶产生菌产酶条件的优化

采用斜面培养和液体发酵培养产甲壳素脱乙酰酶的真菌构巢曲霉,并且研究了产酶条件。结果表明,构巢曲霉的最适产酶条件为：发酵培养基初始pH值为6.5、发酵时间为96h、培养温度为31℃、碳源浓度为2%、氮源浓度为2%、金属离子浓度为0.01mol/L、接种量为6%。     

The effect of lactate addition on the growth of Penicillium camembertii on glutamate

The effect of an additional carbon source, lactate, on Penicillium camembertii growth on glutamate as both carbon and nitrogen sources was examined. Glutamate (and lactate) was present in excess in both media. Throughout the whole culture, similar growth time-courses were recorded on both media, indicating the absence of a lactate effect on growth. During the first part of growth, corresponding to an increasing amount of viable biomass, the rate of glutamate consumption remained high, as well as the related ammonium production, indicating its use as a carbon source in addition to being nitrogen source. The low growth rates recorded during the last part of growth resulted in low glutamate consumption, while lactate consumption continued mainly by a maintenance mechanism for the energy supply. A clear differentiation appeared therefore between the carbon source and the energy source: glutamate was mainly used as C source (and N source) for biosynthesis, while lactate was mainly assimilated for energy supply. Carbon and nitrogen yield examinations confirmed this result. Indeed, the C/N ratio found for P. camembertii cellular material (8.14) was about twice that of glutamate (4.29). From this, about half of the available nitrogen was used for biomass formation during growth on glutamate-lactate based medium, as experimentally confirmed (constant yield nitrogen from biomass on nitrogen from glutamate was found (0.49), while the excess nitrogen was released as ammonium). The constant and close to unit (0.99) yield carbon from CO2 on carbon from lactate, also recorded during growth on glutamate-lactate based medium, confirmed that lactate was mainly used as an energy source.     

Regulation of biosynthesis of vermiculin and vermistatin inPenicillium vermiculatum

Biosynthesis of vermiculin (1) and vermistatin (2) inPenicillium vermiculatum can be controlled by the carbon and nitrogen sources. Glucose and sucrose affect the levels of the two metabolites; cornsteep liquor influences the quality the biosynthesis. The concentrations of Fe³⁺ and Cu²⁺ ions also affect the biosynthesis, the effect being dependent on the type of carbon source utilized. The compounds capable of electron transport generally stimulate the production of1 and2 but do not influence the biosynthesis qualitatively. Translated by Č. Novotny     

Application of mineral support on cephamycin C production in culture using soybean oil as the sole carbon source

A mineral support was used for cephamycin C production in a culture using soybean oil as the sole carbon source. When the support was added into an oil-water system, the soybean oil was emulsified as fine oil droplets, which was observed by a photomicroscope. Mycelia were also twined around the support, which was observed by a scanning electron microscope. That caused the formation of an oil-mycelia complex on or around the support, which provided a larger specific surface area of oil. When 15 g/L of the support was used in a batch culture of Streptomyces sp. P6621 with 50 g/L of soybean oil, the maximum cephamycin C concentration was 2.8 g/L, which was 2.2 times higher than that without the support. This indicates that the mineral support is useful for the culture system using vegetable oil as a carbon source. (c) 1997 John Wiley & Sons, Inc.     

Enzymes of Candida albicans cell-wall lytic system produced by Streptomyces thermodiastaticus

The production of the enzymes of Candida albicans cell-wall lytic system by S. thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest lytic activity was obtained after 18 h of incubation at pH 5.5 and an incubation temperature of 50 degrees C. The carbon source influenced the production of the enzymes of the yeast cell wall lytic system. Maximum lytic activity was obtained when Candida albicans cell-wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml level was the best nitrogen source for the biosynthesis of the enzymes of the yeast lytic system. From all phosphor sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/l) and Tween 80 (0.1%), respectively were found to favour highest enzymes production of the lytic system. The Candida albicans cell-wall lytic system produced by S. thermodiastaticus mainly contained chitinolytic and proteolytic activities.     

Effect of Various Sources of Nitrogen and Carbon on the Biosynthesis of Proteolytic Enzymes in a Culture of Aspergillus awamori 21/96

Effects of various conditions of nitrogen and carbon nutrition on the biosynthesis of proteolytic enzymes in a selected culture of Aspergillus awamori 21/96 were studied. This strain was shown to produce proteolytic enzymes constitutively. In the presence of mineral sources of nitrogen, the synthesis of the enzymes under study was not induced by proteinaceous substrates. Optimum conditions of the enzyme biosynthesis were achieved with casein as a source of nitrogen and starch or dulcitol as a source of carbon (which increased the production of the enzymes by 1.7 and 8 times, respectively). When the cells were grown on starch, their specific activity exceeded control levels by 18 times.     

Organic or mineral nitrogen source during Penicillium camembertii growth on a glucose limited medium

Penicillium camembertii was cultivated on a carbon-limited medium (glucose). Two nitrogen sources were compared, a mineral, ammonium, and an organic nitrogen source, lysine. Among the amino acids convenient nitrogen sources for P. camembertii, lysine was chosen since it cannot be assimilated as a carbon source for cell biosynthesis. During culture on glucose and ammonium, a decline phase immediately followed growth after glucose depletion, since no energy source remained in the medium. On the contrary, on glucose and lysine, a stationary state was recorded after glucose depletion, since lysine was used as the energy supply for cell maintenance, leading to the release of the corresponding carbon as CO₂, while nitrogen from lysine was released as ammonium.     

蛹虫草菌生物合成虫草素的研究进展

蛹虫草Cordyceps militaris是我国传统的药用真菌,虫草素是蛹虫草的主要活性成分,具有抗癌、抗肿瘤、抗病毒等多种生理功能。蛹虫草菌液体发酵是最有希望实现高效生产虫草素的途径,但现阶段生产强度低,亟需应用发酵工程及代谢工程手段提高虫草素产量。文中对液体发酵体系中培养基组分(碳/氮源、前体物质、金属离子等)和培养条件(pH、溶氧量、光照等)对虫草素产量的影响进行了总结,并对虫草素的分离纯化、生物合成基因簇及合成代谢途径进行了阐述,最后探讨了实现虫草素高效生产的关键环节。     

Streptomyces spheroides mutant deprepressed for exoprotease biosynthesis

The effect of carbon, nitrogen and sulfur sources on the biosynthesis of exoproteases was studied with the parent Streptomyces spheroides strain 35 and its mutant M8-2. Addition of a carbon, nitrogen and sulfur source to the medium deficient in one of these elements did not repress the synthesis of exoproteases by the washed mycelium of the mutant as compared to the parent strain. Protein as a sole source of carbon, nitrogen and sulfur had no effect on the biosynthesis of exoproteases by the mutant. In contrast to the parent strain, the biosynthesis of exoproteases in the mutant was not controlled by metabolite repression.