Characterization of glk, a gene coding for glucose kinase of Corynebacterium glutamicum

The glk gene from Corynebacterium glutamicum was isolated by complementation using Escherichia coli ZSC113 (ptsG ptsM glk). We sequenced a total of 3072 bp containing the 969-bp open reading frame encoding glucose kinase (Glk). The glk gene has a deduced molecular mass of 34.2 kDa and contains a typical ATP binding site. Comparison with protein sequences revealed homologies to Glk from Streptomyces coelicolor (43%) and Bacillus megaterium (35%). The glk gene in C. glutamicum was inactivated on the chromosome via single crossover homologous recombination and the resulting glk mutant was characterized. Interestingly, the C. glutamicum glk mutant showed poor growth on rich medium such as LB medium or brain heart infusion medium in the presence or absence of glucose, fructose, maltose or sucrose as the sole carbon source. Growth yield was reduced significantly when maltose was used as the sole carbon source using minimal medium. The growth defect of glk mutant on rich medium was complemented by a plasmid-encoded glk gene. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression, and it was found that glk was expressed constitutively under all tested conditions with different carbon sources.     

Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth.     

Formation and distribution of free amino acids in Cunninghamella elegans mycelium during growth on media with glucose and dodecane]

The growth, assimilation of organic nitrogen compounds, and production and distribution of free amino acids were studied during the growth of Cunninghamella elegans (--) 1204 on the defined medium 12 containing glucose and dodecane and without an additional carbon source (control). The culture utilized all leucine, glycine, and asparagine after three days of the growth, irrespective of the source of carbon in the medium. Glutamic acid was assimilated only in the presence of glucose. Some 15 free amino acids and 2 amides were detected in the cells, and also some correlations in the composition of the pool in the presence of different sources of carbon in the medium. The substitution of dodecane for glucose decelerates metabolism of free intracellular amino acids and stimulates their accumulation in the cells, especially if there is a deficiency of carbon sources: the overall content of amino acids and amides on the medium with glucose is from 2.2 to 4.1 mg/g; with dodecane, from 9.8 to 16.5 mg/g; in the control, from 16.4 to 25.8 mg/g.     

Growth characteristics and metabolic flux analysis of Candida milleri

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.     

不同碳源和氮源对金针菇降解木质纤维素酶活性的影响

以3株栽培的金针菇Flammulina velutipes为材料,研究它们在玉米芯和棉子壳以及不同碳源、氮源培养条件下纤维素、半纤维素和木质素降解酶活性的规律。结果表明,不同金针菇菌株的羧甲基纤维素酶、木聚糖酶和漆酶活力显著不同（P<0.001）,同时,培养条件对羧甲基纤维素酶、木聚糖酶和漆酶的活力都有显著影响（P<0.001）。在简单碳源存在的条件下,金针菇的羧甲基纤维素酶和木聚糖酶活力远远低于复杂碳源培养基（P<0.05）。全营养培养基上生长的金针菇的羧甲基纤维素酶和木聚糖酶活力低于缺乏碳源和氮源的培养基（P<0.05）。漆酶活力在无简单氮源培养基上低于全培养基（P<0.05）和无葡萄糖培养基（P<0.05）,即复杂碳源和氮源培养基上的漆酶活力低于简单碳源和氮源培养基（P<0.05）。     

Influence of sugars on blue light-induced synthesis of chlorophyll in cultured plant cells

In supension cultured tobacco cells only blue light induces and maintains chlorophyll synthesis if the liquid nutrient medium is supplemented with sucrose. The yield per gram fresh weight is closely correlated with the energy fluence rate of blue light, but not with the initial amount of sucrose added to the medium (3–12 g/l). The uptake of sucrose by the cells proceeds with a constant rate over the growth period independently of the initial amount leading within 10–25 days to sucrose-free media. Under these conditions the cells continue to synthesize chlorophyll for about 10 days. This limitation is overcome by adding sucrose to the medium at equal time intervals thus establishing a constant sugar level beyond the growth period. In contrast, glucose as carbon source cannot adequately replace sucrose in inducing and maintaining blue light-induced chlorophyll synthesis. Depending on the initial amount (3–10 g/l) this sugar is rapidly disappearing from the medium within 1–5 days after inoculation of the cells. It apparently serves as a preferential source of energy and carbon skeletons thus suppressing chlorophyll synthesis. On the other hand, glucose combined with sucrose in the medium brings about the characteristic induction and accumulation of chlorophyll in blue light which is observed with sucrose as the sole carbon source.Abbreviation EFR energy fluence rate - FW fresh weight - MS-medium Murashige-Skoog medium (Murashige and Skoog 1962)     

Growth yields of methanotrophs

Summary The growth yield ofMethylococcus capsulatus (Bath) on methane was dependent on the availability of copper in the growth medium. In nitrate mineral salts medium the carbon conversion efficiency increased by 38%, concomitant with the transition from soluble to particulate methane monooxygenase, after transfer from low to high copper medium. An increase in growth efficiency was also observed with ammonia as nitrogen source but not when methanol replaced methane as carbon source. The high growth efficiency is attributed to a reduced NADH requirement for methane oxidation. This could only arise if methanol dehydrogenase was capable of electron transfer, either directly or indirectly to the particulate methane monooxygenase (MMO). The carbon conversion efficiency from methanol with nitrate as nitrogen source was as high as theoretically predicted. It is suggested that the previously low yields of methanotrophs grown on methanol resulted from the use, as nitrogen source, of ammonia which was oxidised by the MMO still present under these growth conditions. The term ‘methanotroph’ is used throughout to distinguish those organisms capable of growth on methane from ‘methylotrophs’ capable of growth on reduced C, compounds other than methane     

Assessment of the production of 13C labeled compounds from phototrophic microalgae at laboratory scale

An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and stripping with N(2). The inorganic carbon of the supernatant was recovered also by acidification and subsequent stripping with N(2). The operating conditions of this step were optimized for gas flow rate and type of contactor. A carbon recovery step for the depleted biomass was designed based on the catalytic oxidation to CO(2) using CuO (10 wt.%) as catalyst with an oxygen enriched atmosphere (80% O(2) partial pressure). In this way, the carbon losses reduced an 80.2% and the efficiency of the conversion of carbon in EPA was increased to 19.5%, which is close to the theoretical maximum. Further increase in 13CO(2) use efficiency is only possible by additionally recovering other labeled by-products present in the biomass: proteins, carbohydrates, lipids, and pigments.     

碳源影响水稻根际假单胞菌PA1201藤黄绿菌素的生物合成

【背景】假单胞菌PA1201是一株水稻根际促生菌,其产生的次生代谢物藤黄绿菌素(pyoluteorin,Plt)能够有效抑制多种植物病原真菌和细菌的生长,但在常规培养条件下Plt产量极低。【目的】研究碳源对Plt生物合成的影响,为提高Plt的产量以及应用提供理论基础。【方法】将基本培养基(minimal medium,MM)中甘露醇替换为不同的碳源及碳源组合作为PA1201的培养基,生长过程中不同时间点取样提取Plt,利用高效液相色谱(HPLC)法分析Plt的产量变化。【结果】建立了基于HPLC定性和定量检测Plt的方法;比较了PA1201菌株在不同培养基中菌株生长和Plt的产量,发现果糖和甘露醇促进Plt生物合成;果糖和甘露醇对Plt生物合成没有增效作用;在含有甘露醇或果糖作为唯一碳源的培养基中,添加葡萄糖或琥珀酸抑制Plt生物合成。【结论】果糖和甘露醇促进水稻根际假单胞菌PA1201合成藤黄绿菌素,这为提高藤黄绿菌素的生物合成效率和促进藤黄绿菌素的应用奠定了基础。     

Penicillinamidohydrolase inEscherichia coli

Synthesis of penicillinamidohydrolase (penicillin acylase, EC 3.5.1.11) in Escherichia coli is subjected to the absolute catabolite repression by glucose and partial repression by acetate. Both types of catabolite repression of synthesis of the enzyme in Escherichia coli are substantially influenced by cyclic 3',5'-adenosinemonophosphate (cAMP). Growth diauxie in a mixed medium containing glucose and phenylacetic acid serving as carbon and energy sources is overcome by cAMP. cAMP does not influence the basal rate of the enzyme synthesis (without the inducer). Derepression of synthesis of penicillinamidohydrolase by cAMP in a medium with glucose and inducer (phenylacetic acid) is associated with utilization of the inducer, due probably to derepression of other enzymes responsible for degradation of phenylacetic acid. Lactate can serve as a "catabolically neutral" source of carbon suitable for the maximum production of penicillinamidohydrolase. The gratuitous induction of the enzyme synthesis in a medium with lactate as the carbon and energy source and with phenylacetic acid is not influenced by cAMP; however, cAMP overcomes completely the absolute catabolite repression of the enzyme synthesis by glucose.     

Growth inhibition of Proteus mirabilis by cyclic adenosine 3''-5''-monophosphate.

Growth of Proteus mirabilis on a synthetic agar medium containing either glycerol, galactose, or trehalose as the sole source is inhibited by 5 mM cyclic adenosine 3',5'-monophosphate (cAMP). Inhibition on an agar medium is evident as loss of viability, but in broth cAMP only slightly inhibits growth rate. Inhibition is associated with the accumulation of methylglyoxal in the medium. A nonswarming mutant of P. mirabilis is not inhibited by cAMP on either of the three carbon sources, but it is sensitive to exogenous methylglyoxal.     

Isolation and identification of Sphingomonas sp. that yields tert-octylphenol monoethoxylate under aerobic conditions

Topsoil samples were collected from eight golf courses in Yamaguchi Prefecture, Japan, and enrichment cultures were carried out with a basal-salt medium containing 0.2% 4-tert-octylphenol polyethoxylate (OPPEO) as sole carbon source. OPPEO-degrading activity was detected in one of the samples, from which a strain of OPPEO-degrading bacterium was isolated. The isolated bacterium grew on a nutritionally enriched medium (NE medium) containing 0.2% OPPEO as sole carbon source, and accumulated 4-tert-octylphenol diethoxylate (OP2EO) (63%), 4-tert-octylphenol triethoxylate (OP3EO) (14%), and 4-tert-octylphenol monoethoxylate (OP1EO) (2%) after 7 d cultivation under aerobic conditions. The addition of clay mineral (vermiculite) to the medium accelerated the degradation of OP2EO (40%) and OP3EO (4%) to OP1EO (23%). This is the first report about bacteria that can degrade OPPEO to OP1EO under aerobic conditions. The strain was identified as Sphingomonas macrogoltabidus, based on the homology of a 16S rDNA sequence.     

Sources of NADPH in yeast vary with carbon source

Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984-13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1Delta ald6Delta mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1Delta idp2Delta mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1Delta ald6Delta mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Delta ald6Delta mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1Delta ald6Delta mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP+ rise dramatically in the zwf1Delta idp2Delta mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Pyruvate carboxylase deficiency in yeast: a mutant affecting the interaction between the glyoxylate and Krebs cycles

A single-gene nuclear mutant has been isolated in Saccharomyces cerevisiae which cannot grow on minimal medium supplemented with ethanol, acetate, pyruvate, aspartate, or oxaloacetate as sole carbon sources. It will grow on complete medium with these carbon sources, and on minimal medium with dextrose as carbon source. The only supplement which will permit growth on minimal medium with ethanol or pyruvate is aspartate, so the mutant is an aspartate auxotroph when grown on these nonfermentable substrates. It exhibits enhanced levels of phosphoenolpyruvate carboxykinase (EC 4.1.1.49) when grown on dextrose. The mutant can survive as an alcohol dehydrogenase-negative, indicating that the defect is not in the Krebs Cycle or in electron transport. When grown on pyruvate, it produces two to three times as much free alanine and half as much aspartate plus asparagine as the wild type. Two different assays show that the mutant phenotype is due to a deficiency of pyruvate carboxylase (EC 6.4.1.1), an important anaplerotic enzyme. Inferences that can be drawn from the characteristics of this mutant include (a) the glyoxylate cycle is probably located entirely outside the mitochondria, (b) the inner mitochondrial membrane appears to be impermeable to oxaloacetate, and (c) a succinate-malate exchange across the inner mitochondrial membrane connects the glyoxylate and Krebs cycles when yeast is grown on minimal medium with ethanol as a sole carbon source.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> high-cell-density culture: carbon mass balances and release of outer membrane components

Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO(2), and acetate was approximately 90% at the end of the culture (25 h, 27 g L(-1) dw). The missing carbon remained as soluble organic compounds in the medium. Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at approximately 1 g L(-1)) contributed to 63% of the extracellular carbon. The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture. Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred. Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the extracellular carbon mass. The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.     

汉森酵母表达载体的构建和人血管生成抑制素基因的表达

汉森酵母(H.polymorpha)是一类能以甲醇为唯一碳源和能源的甲基营养酵母,具有高表达外源基因、易于高密度发酵和产业化的特点。应用PCR技术扩增汉森酵母甲醇氧化酶(Methanol oxidase MOX)基因启动子和转录终止序列,并与汉森酵母Leu基因(Hpleu2)和人血管生成抑制素基因一起重组进大肠杆菌质粒pSP72,构建了整合型表达载体pSMA17,采用LiAc法将pSMA17转入汉森酵母A16(leu),筛选出阳性转化子H.polymorpha A16(pSMA17)。转化子在YPGE培养基中培养至对数生长后期,用甲醇进行诱导表达。ELISA和SDSPAGE分析结果证明人血管生成抑制素已获表达,表达产物分泌至培养基中。Western blot结果显示重组的人血管生成抑制素能与抗人纤溶酶原抗血清特异结合,具有免疫原性。     

Selective removal of molybdenum traces from growth media of N2-fixing bacteria

A new method for the selective removal of traces of molybdenum from growth media of N2-fixing bacteria (Rhodobacter capsulatus and Klebsiella pneumoniae) was developed. This method is based on the filtration of nutrient solutions through a layer of activated carbon (pulverized charcoal). The adsorption of Mo (molybdate) to activated carbon was optimal if a charcoal suspension (50 g/liter) was degassed by boiling before use and if the pH of the solutions, which had to be purified, was adjusted to values between 1.5 and 4. In this pH region no or only negligible amounts of other metal ions were adsorbed. The activated carbon method was compared with other Mo-eliminating procedures, including 8-hydroxyquinoline/dichloromethane extraction, Chelex 100 chromatography, and treatment with Mo-starved Azotobacter vinelandii cells. The activated carbon filtration appeared to be the most effective, specific, and rapid method. Whereas the untreated Rhodobacter growth medium was contaminated with 1.2 ppb Mo, as analyzed by inductively coupled plasma mass spectrometry (ICP-MS), the activated carbon-treated medium was below the ICP-MS detection limit (less than 0.05 ppb). A similarly effective removal of Mo impurities was obtained by the Azotobacter treatment. Even at low optical densities (2-5 at 436 nm) Mo traces were removed very rapidly within 10-15 min. However, because the Mo uptake/Mo adsorption capacity of A. vinelandii depended on freshly cultivated cells and on the growth phase at which the cells were harvested, this microbiological method was generally more time-consuming and less reproducible than the activated carbon method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.