5-aminolevulinic acid biosynthesis in Escherichia coli coexpressing NADP-dependent malic enzyme and 5-aminolevulinate synthase

5-aminolevulinate (ALA) synthase (E.C. 2.3.1.37), which mediates the pyridoxal phosphate-dependent condensation of glycine and succinyl-CoA, encoded by the Rhodobacter sphaeroides hemA gene, enables Escherichia coli strains to produce ALA at a low level. To study the effect of the enhanced C4 metabolism of E. coli on ALA biosynthesis, NADP-dependent malic enzyme (maeB, E.C. 1.1.1.40) was coexpressed with ALA synthase in E. coli. The concentration of ALA was two times greater in cells coexpressing maeB and hemA than in cells expressing hemA alone under anaerobic conditions with medium containing glucose and glycine. Enhanced ALA synthase activity via coupled expression of hemA and maeB may lead to metabolic engineering of E. coli capable of large-scale ALA production.     

Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.     

重组Rhodobacter sphaeroides hemA表达的调控

RhodobactersphaeroideshemA编码5氨基乙酰丙酸合酶(ALAS),催化磷酸吡哆醛依赖性琥珀酰CoA和甘氨酸缩合成ALA.将R.spaeroideshemA导入E.coli进行表达,当hemA具有与lac启动子相同的转录方向时,ALAS有活性.lac启动子与hemA之间的距离会影响ALAS在不同培养基上的表达.E.coli宿主菌对ALAS表达、ALA产量有显著影响,在实验所用6种菌株中,E.coliDH1是最佳宿主菌(P<0.05).ALAS表达还与碳源有关,琥珀酸为碳源时,重组ALAS活性最高(P<0.05),以乳酸为碳源时,ALAS活性很低.重组ALAS活性也受培养基pH值影响,pH6.5时,活性最高(P<0.05).     

Phasic changes in reproductive potential, intracellular polyamines and antilysozyme activity in Escherichia coli in periodic culture

Growth-phase associated changes in and relationships between the specific growth rate (mu) characterizing the reproductive capacity of the cells, the contents of intracellular biogenic polyamines (BPA), such as putrescine (P), cadaverine (C), and spermidine (S), and antilysozyme activity (ALA) were studied in 37 strains of Escherichia coli grown in batch culture on solid medium. A decrease in mu upon the transition of the culture to the stationary growth phase was accompanied by a decrease in the pool of free BPA, mainly P and C, and by the appearance of ALA. The interrelations between the parameters studied were described as a complex of direct and negative correlations; the combination of low initial P and C contents, reduced P/S and C/S ratios, and a high level of ALA was designated as a factor of slight inhibition of E. coli reproduction. It is argued that BPA and ALA are integrated in a system controlling both the metabolism and stability of peptidoglycan in E. coli.     

Effect of a polyoxydonium immunoregulator on the biological properties of microorganisms

The effect of the synthetic immunomodulator polyoxydonium (PO) on some biological properties of pathogenic bacteria (Shigella flexneri, Salmonella enteritidis), opportunistic bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacteroides fragilis, Peptostreptococcus anaerobius, Prevotella melaninogenica, Propionibacterium propionicum, Clostridium difficile) and fungi (Candida albicans), isolated during enteric infections, enteric dysbiosis, pyoinflammatory diseases, was evaluated in a number of in vitro experiments. The study revealed that the recommended therapeutic concentration of PO decreased antilysozyme activity (ALA) and the anticomplement activity in Klebsiella, Shigella, Propionibacterium, Clostridium, bacteroids, fungi of the genus Candida, but increased ALA in nonhemolytic Escherichia. Under the action of PO an increased sensitivity of the microorganisms under study to definite antibiotics of the lincosamide, fluoroquinolon, carbapenem and cephalosporin groups was noted. The data obtained in this study reveal one of the possible mechanisms of the corrective action of PO on the microbiocenosis of the intestine in dysbiosis, enteric infections and pyoinflammatory diseases.     

Impact of indigenous microorganisms on Escherichia coli O157:H7 growth in cured compost

Both autoclaving and dry-heat treatments were applied to dairy manure-based compost to achieve target populations of indigenous microorganisms. A 3 strain-mixture of Escherichia coli O157:H7 of ca. 2 log CFU/g was inoculated into acclimated autoclaved compost (AAC) and dry heat-treated compost (DHTC) with different moistures, and stored at 8, 22, or 30 °C. Only selected groups of microorganisms grew in AAC during acclimation, whereas the relative ratio of each group of microorganisms was maintained in DHTC after heat treatment. E. coli O157:H7 grew more in AAC than DHTC in the presence of same level of indigenous mesophiles. However, control compost (no heat treatment) did not support E. coli O157:H7 growth. Our results revealed that both the type and population of indigenous microorganisms is critical for suppressing E. coli O157:H7 growth in compost, and dry-heat treatment can result in a compost product which resembles cured compost with different levels of indigenous microorganisms.     

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.     

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).     

5-Aminolevulinic acid synthesis in Escherichia coli.

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.     

Antimicrobial action of Lentinus edodes juice on human microflora

The action of the juice of Shiitake mushroom (L. edodes) on pathogenic and opportunistic microorganisms, detected in cases of considerable dysbiotic changes (Escherichia coli O-114, Staphylococcus aureus, Enterococcus faecalis, Candida albicans), as well as on some bacterial eubiotic producer strains (Escherichia coli M-17, Bifidobacterium spp., Lactobacillus spp.). The juice of this mushroom at a concentration of 5% from the volume of the nutrient medium was found to produce a pronounced antimicrobial effect with respect to C. albicans, S. aureus, E. faecalis, E. coli O-114 and to stimulate the growth of E. coli M-17. Bifidobacteria and lactobacteria exhibited resistance to the action of L. edodes juice.     

Evidence for a magnesium pump in Bacillus cereus T

Unlike Escherichia coli, Bacillus cereus T appears to accumulate Mg(2+) in its cell sap against a concentration gradient. Over a range of Mg(2+) in the growth medium from 5 x 10(-5) to 1.35 x 10(-2)m, the concentration of Mg(2+) in the cell sap of B. cereus T was maintained at about 6 x 10(-3)m, and ribosome-bound Mg(2+) and spermidine, as well as the spermidine concentration in the cell sap, appear to be unaffected by the concentration of Mg(2+) in the growth medium. Inhibition of growth of E. coli by streptomycin is progressively reversed by increasing the concentration of Mg(2+) in the growth medium above 5 mm. The finding that similar increases of Mg(2+) in the growth medium did not reverse the inhibition of B. cereus T is also consistent with the conclusion that B. cereus T, unlike E. coli, accumulates Mg(2+) to a constant concentration in its cell sap.     

Behavior of a hybrid F' ts114 lac+, his+ factor (F42-400) in Klebsiella pneumoniae M5a1.

Episome F' ts114 lac+, his+ (F42-400) was transferred from Salmonella typhimurium to Klebsiella pneumoniae. From the progeny, a strain of K. pneumoniae able to retransfer the episome was obtained. The His+ phenotype in this strain is temperature sensitive. Escherichia coli female-specific phages phiII, W31, and T3 were shown to plate on K. pneumoniae. From phiII we obtained two derivatives; phiIIK, which plates only on K. pneumoniae, and phiIIE, which plates only on E. coli. Growth of phages T3 and phiIIK was inhibited by F42-400 in K. pneumoniae. Growth in presence of acridine orange in a defined medium at 40 C resulted in a high level of curing. The frequency of His+ cells after growth in acridine orange at 40 C was 0.001%. An extensive search to detect chromosome mobilization by F42-400 in K. pneumoniae, under different experimental conditions, was negative. We cannot exclude the possibility that the low transfer efficiencies prevented our detection of chromosome mobilization. A search among temperature-resistant, acridine orange-curing-resistant, or galactose-resistant derivatives of the K. pneumoniae donor strain failed to reveal any chromosome transfer. Our failure to detect Hfr's may be a result of: (i) the peculiarity of episome F42-400, (ii) the peculiarity of K. pneumoniae chromosome, or (iii) low transfer efficiency. K. pneumoniae-modified F42-400 and phage 424 were restricted by E. Coli K-12. E. coli K-12-modified episome F42-400 and phage 424 were restricted by K. pneumoniae. E. coli C failed to restrict F42-400 modified with K. pneumoniae specificity. The ability of K. pneumoniae to accept F42-400 is less, by about a factor of 50, than that of E. coli C. As an explanation for the differences in the behavior of E. coli C and K. pneumoniae in ability to receive F42-400 it was suggested that recipient bacteria have specific sites for interaction with the F-pilus tip; these are present in E. Coli C, leading to high transfer efficiency, whereas they may not be present (or if present, are not accessible) in K. pneumoniae, leading to low transfer efficiency.     

Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.     

The effect of inulin on the biological properties of enterobacteria

The influence of inulin on some biological properties of Enterobacteriaceae, both pathogenic (Salmonella) and opportunistic (Klebsiella, Escherichia coli) was experimentally determined in vitro. The study revealed that inulin stimulated the production of colicin and suppressed the hemolytic activity in E. coli. The effect produced by inulin on the antilysozyme activity (ALA) of Enterobacteriaceae was different: in S. typhimurium ALA was decreased and in most K. pneumoniae strains the expression of this sign was enhanced.     

Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).     

Reliability of the disinfecting action of pulse electrical discharges in water treatment]

The irreversibility of the damaging action of pulse electric discharges (PED) on microorganisms in the process of the disinfection of water was studied. The experiments with standard E. coli culture showed that bacterial cells remaining alive after being subjected to the action of PED eventually died the sooner, the higher the temperature of the medium was. In any of the "blind passages" made within 7 days no growth of the test microorganism was observed. The use of enriched fluid media and 10-day incubation did not ensure the preservation of proliferation ability of the cells after the action of PED with the parameters used in the experiment.     

Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli.

Several mutants of Escherichia coli that had lost their ability to synthesize delta-aminolevulinic acid (ALA) via the C5 pathway were isolated. Their defective loci were classified into two groups, AlaA- and AlaB-. The genes that complemented these mutations were cloned. Nucleotide sequencing indicated that the gene that complemented AlaA- was identical to hemL which is located at 4 min on the E. coli chromosome and encodes glutamate 1-semialdehyde aminotransferase. The gene complementing AlaB- contained an open reading frame (ORF) encoding a polypeptide of 207 amino acids that was found to be a new gene involved in the synthesis of ALA via the C5 pathway. Thus, we designated the gene hemM. The hemM gene was adjacent to hemA that is located at 27 min and previously thought to encode glutamyl-tRNA dehydrogenase. However, we found that hemA complemented both the AlaA- (hemL) and AlaB- (hemM) mutants defective in the C5 pathway although the transformants showed small colonies on the selective medium without ALA. These results suggest that hemA is not involved in the C5 pathway, but controls a second, minor pathway for the synthesis of ALA.     

The effects of Korean propolis against foodborne pathogens and transmission electron microscopic examination

This study was performed to evaluate the effects of Korean propolis against foodborne pathogens and spores of Bacillus cereus and to investigate the antimicrobial activity against B. cereus structure by transmission electron microscopy (TEM). The antimicrobial effects of the Korean propolis were tested against foodborne pathogens including Gram-positive (B. cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Escherichia coli and Pseudomonas fluorescence) bacteria by agar diffusion assay. Gram-positive bacteria were more sensitive than were Gram-negative bacteria. The vegetative cells of B. cereus were the most sensitive among the pathogens tested with minimum inhibitory concentration (MIC) of 0.036 mg/μl of propolis on agar medium. Based on MIC, sensitivity of vegetative cells of B. cereus and its spores was tested in a nutrient broth with different concentrations of propolis at 37°C. In liquid broth, treatment with 1.8 mg/ml propolis showed bactericidal effect against B. cereus. B. cereus vegetative cells exposed to 7.2mg/ml of propolis lost their viability within 20 min. Against spores of B. cereus, propolis inhibited germination of spores up to 30 hours, compared to control at higher concentration than vegetative cells yet acted sporostatically. The bactericidal and sporostatic action of propolis were dependent on the concentration of propolis used and treatment time. Electron microscopic investigation of propolis-treated B. cereus revealed substantial structural damage at the cellular level and irreversible cell membrane rupture at a number of locations with the apparent leakage of intracellular contents. The antimicrobial effect of propolis in this study suggests potential use of propolis in foods.     

大豆ω-3脂肪酸脱氢酶基因GmFAD3C在酿酒酵母中的表达

α亚麻酸(ALA)被称为必需脂肪酸,对人体有一系列的保健作用。ω-3脂肪酸脱氢酶(FAD)催化亚油酸(LA)生成ALA。大豆种子油中ALA含量较高,为了研究大豆ω3FAD的功能,用RTPCR方法从大豆未成熟种子中扩增出GmFAD3C的cDNA,克隆到酵母表达载体p416中,并用醋酸锂法转化酿酒酵母营养缺陷型K601,经筛选鉴定,得到阳性克隆。气相色谱分析脂肪酸成分,发现工程菌产生了新的脂肪成分ALA,含量占总脂肪酸的3.1%,LA含量与对照相比相应地下降,证明该基因编码的蛋白具有催化18碳多不饱和脂肪酸(PUFA)底物LA在Δ15位脱氢生成ALA的ω3FAD功能,首次实现大豆ω-3脂肪酸脱氢酶基因在酿酒酵母K601p416系统中的表达,建立了一种新的高效低成本的FAD酵母表达系统。     

Optimization of extracellular 5-aminolevulinic acid production from Escherichia coli transformed with ALA synthase gene of Bradyrhizobium japonicum

Extracellular accumulation of 5-aminolevulinic acid (ALA) by an E. coli overexpressing ALA synthase (ALAS) was achieved by inserting a hemA gene from Bradyrhizobium japonicum and expressed under the control of T7 promoter. At pH 7.0 extracellular ALA reached up to 15 mM in a jar fermenter with an addition of glycine (30 mM) and succinate (90 mM) in the medium. ALA accumulation was increased to 20 mM by adding levulinic acid (30 mM) to the cultures.