Studies on the Utilization of Hydrocarbons by Microorganisms

It is well known that a good many microorganisms can utilize hydrocarbons as a sole carbon source. We have as yt little information, however, as to what kinds of intermediate or end products are produced by the microbial dissimilation of hydrocarbons. Above all, the formation of amino acids from hydrocarbons has not been reported. We have isolated many strains of microorganisms from soil samples by selective culture techniques in a medium containing kerosene and mineral salts, in order to examine if the products of economical value such as amino acids, organic acids, fatty acids, steroids, nucleic acids and their related compounds, can be produced by those microorganisms. Shaking cultures of those microorganisms were carried out in the medium containing 3.5% kerosene, 3.5% liquid paraffin and mineral salts (K₂HPO₄ 0.25%; MgSO₄·7H₂O 0.1%; NH₄MO₈ 0.5% or urea 0.3%); we found that 55 strains out of 127 strains tested produced amino acids in their broths, after 4 days cultivation at 26.5°C. The kinds and maximum yields of amino acids were as follows: alanine 50mg/l, aspartic acid 23mg/l, glutamic acid 485 mg/1, glycine 8.5mg/l, isoleucine 15 mg/1, lysine 23.9 mg/1, and valine 38 mg/1.     

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.     

亚热带森林土壤微生物生物量及群落功能特征的城乡梯度变化

土壤微生物在土壤养分循环以及生物地球化学循环中起着重要作用,土壤微生物对环境变化响应灵敏.为研究城乡梯度环境变化对亚热带森林土壤微生物的影响,本研究选取合肥市大蜀山国家森林公园(城市林)、紫蓬山国家森林公园(远郊林)、六安市万佛山(乡村自然林)为样地,分析比较其微生物生物量碳(MBC)、微生物生物量氮(MBN)及微生物...     

The impact of dissolved amino acids on protein and cellulose degradation in stream waters

Dissolved amino acids represent a significant carbon and nitrogen source for microorganisms in stream water environments, and may be utilized in preference to protein bound nitrogen in decomposing organic matter. Consequently, in streams with sufficiently high concentrations of dissolved amino acids, depolymerisation of nitrogen compounds may become delayed. This possibility was investigated in stream water samples incubated with ¹⁴C-labelled albumin and cellulose in presence of the indigenous microorganisms at different concentrations of dissolved amino acids.The experiments demonstrated at an average between 15 and 25% lower degradation of the compounds during a ten days incubation at an initial amino acid concentration of 1 mg l^–1. The reduction was most clearly expressed in a nutrient-poor stream water compared with a nutrient-rich. The kinetics of the degradation were most appropriately described by a first-order model, that is, the rate of transformation of the macromolecules was independent of the total number of bacteria in the water. The mechanism suggested for the retardation of macromolecules is a superproportional utilization of the dissolved amino acids at high concentrations, a phenomenon that can cause accumulation of slowly decomposing macromolecules in sediments affected by residual wastewater.     

Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and its effects on growth and dechlorination activity

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with (13)C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.     

Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

Enhanced Activity of Pyruvic Kinase in the Potassium-Deficient Wheat Leaves

Isolation and screening tests were carried out to obtain microorganisms capable of producing amino acids from pentoses and hexoses directly in the presence of nitrogen sources and inorganic sails.

Three strains having exceedingly high amino acids producing ability were obtained. One of them produces remarkably l-glutamic acid and alanine and the other two have high productivity of alanine.

As a result of the taxonomical studies, all of these strains were found to belong to the Genus Brevibacterium. Moreover, there are no species which conform to these strains described in Bergey’s Manual of Determinative Bacteriology 7th Ed., so the authors decided them to be new species.     

Microbial metabolism of 2-methyl amino acids

Eighty-nine microorganisms were isolated that were able to use 2-methyl amino acids and related compounds as the sole source of nitrogen. All of these cultures produced low levels of ammonia in culture supernatant solutions None was capable of fixing nitrogen gas. Whole-cell and cell-free-extract experiments showed that ammonia was not released directly from the 2-methyl amino acids. All of these strains except those isolated with 2-methylserine as a nitrogen source appeared to metabolize 2-methyl amino compounds by a single enzymatic reaction involving simultaneous decarboxylation and transamination. Pyruvate served as an acceptor for the transamination with the concomitant formation of alanine. The strains utilizing 2-methylserine produced a specific 2-methylserine transhydroxymethylase.     

Protein synthesis in tomato-fruit locule tissue: Incorporation of amino acids into protein by aseptic cell-free systems

1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating (14)C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20-45mumumoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10-20mumumoles of amino acid/mg. of protein; 40-150mumug. of carbon/mg. of protein was incorporated in 10min. from (14)C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10-20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

黑水虻幼虫氨基酸及其微量元素分析

黑水虻具有较高的营养价值,是一种重要的环境友好型资源昆虫.本研究采用凯氏定氮法、茚三酮柱后衍生离子交换色谱法和微波消解-电感耦合等离子质谱法(ICP-MS)等多种分析技术,测定了厨余垃圾饲喂的黑水虻幼虫蛋白质、氨基酸和微量元素的组成及含量.研究结果表明,黑水虻幼虫中蛋白质的含量为40.60 g/100 g.在测定的16...     

鸡内金中氨基酸及营养元素含量的测定

采用日立 835 - 5 0型氨基酸自动分析仪和岛津Z - 80 0 0型原子吸收分光光计。测定了鸡内金中氨基酸和营养元素的含量 ,结果表明 :鸡内金中营养元素钾、钙、镁、铜、锌、铁、锰含量分别为 36 9.5 0、12 0 .5 6、470 .77、31.89、32 .42、5 5 9.6 4、8.11mg/kg ;氨基酸总量为 86 .92 4% ,必需氨基酸占氨基酸总量的 30 .2 6 4%。     

Extracellular accumulation of a new amino acid, O-2-hydroxypropylhomoserine, from 1,2-propanediol by flavobacterium rigense.

During an investigation of microorganisms utilizing petrochemicals, a strain identified as Flavobacterium rigense was found to accumulate a new amino acid in a medium containing 1,2-propanediol as the sole carbon source. Cultural conditions for the accumulation of the product were investigated, and as a result, the yield was increased to 2.8 mg/ml after a 5-day incubation in a medium containing 8% 1,2-propanediol. The pure amino acid was isolated, and its structure was investigated. Elemental analysis and infrared, nuclear magnetic resonance, and mass spectral analyses indicated that the amino acids is O-2-hydroxypropylhomoserine.     

Isolation,characterization and comparison of bacteria from swine faeces and manure storage pits

Storage of swine manure is associated with the microbiological production of a variety of odorous chemicals including ammonia, organic acids and alcohols, and sulphides. Although largely the product of microbiological activity, little is known about the microorganisms present in swine manure. In order to gain a better understanding of the types and activities of the microorganisms present, representative strains of microorganisms were isolated from faeces and stored manure slurry, identified, and physiologically characterized. For swine manure slurry samples, total anaerobe colony counts were greatest when a non-selective, habitat simulating medium containing clarified swine manure slurry was used whereas the highest counts for faecal anaerobes were obtained on rumen fluid containing medium. Faecal and slurry samples were also plated onto the appropriate medium containing the antibiotics tetracycline, erythromycin and tylosin (10 micro g ml-1, individually) and the proportional counts of organisms capable of growing in the presence of these antibiotics determined. Randomly selected isolates from the highest dilutions were identified by 16 s rDNA sequence analysis, and selected physiological characteristics were determined. The results of these examinations indicate that the predominant culturable microorganisms from these environments are obligately anaerobic, low mol percentage G + C Gram positive bacteria (Firmicutes) who are members of Clostridial, Eubacterial, and Lactobacillus/Streptococcus phylogenetic groups. Isolates similar to Sporomusa and Flexibacter/Cytophaga/Bacteroides (CFB or Bacteroidetes) groups were also obtained. Although similar overall, faecal and slurry samples differed in bacterial composition. Manure slurry samples were dominated by organisms similar to Clostridium coccoides and Enterococcus species whereas the distribution of species present in faeces appeared much broader. Whereas most of the pure cultures could be assigned to known phylogenetic groupings, few could be identified as known species. Examination of some growth and physiological characteristics of faecal and slurry isolates showed these to be primarily carbohydrate fermenters, although some were able to ferment lactate and amino acids. When the ability of manure and faecal isolates to ferment protein, peptides and amino acids was examined, a relatively small percentage of these were able to do so and most of these fermented carbohydrates in addition to the amino acid sources provided. The predominant amino acid fermenters were most closely related to C. coccoides and C. botulinum, but representatives of the Bacteroides, Staphylococcus, Enterococcus and other phylogenetic groups were also found. The results reported here are compared with those obtained from clone libraries prepared from the same environmental samples.     

Extracellular accumulation of a new amino acid, O-2-hydroxypropylhomoserine, from 1,2-propanediol by flavobacterium rigense.

During an investigation of microorganisms utilizing petrochemicals, a strain identified as Flavobacterium rigense was found to accumulate a new amino acid in a medium containing 1,2-propanediol as the sole carbon source. Cultural conditions for the accumulation of the product were investigated, and as a result, the yield was increased to 2.8 mg/ml after a 5-day incubation in a medium containing 8% 1,2-propanediol. The pure amino acid was isolated, and its structure was investigated. Elemental analysis and infrared, nuclear magnetic resonance, and mass spectral analyses indicated that the amino acids is O-2-hydroxypropylhomoserine.     

Partial purification of alanine carrier from rabbit small intestine brush border membrane and its functional reconstitution into proteoliposomes

An alanine transport carrier was partially purified from brush border membranes of rabbit small intestine. The alanine carrier activity was not solubilized with 0.4% deoxycholate but recovered in the detergent-insoluble fraction. The detergent-insoluble proteins were reconstituted into proteoliposomes with soybean phospholipids. The reconstituted proteoliposomes were capable of uptake of alanine driven by an electrochemical potential of Na+. The initial rate of alanine uptake into the proteoliposomes was 90 pmoles/mg protein/sec, which was 15-fold higher than that observed with the native membrane vesicles. The uptake of alanine was effectively suppressed by various neutral amino acids but not by either cationic or anionic amino acids.     

Effect of Chemical Structure on the Biodegradability of Aliphatic Acids and Alcohols

Sewage microorganisms readily degraded unsubstituted aliphatic acids, but the rate of decomposition was much slower with substituted acids as substrates. The type, number, and position of the substituents governed the rate of the oxidation. A single halogen, particularly if on the α-carbon, decreased the rate of biodegradation, but the dihalogenated compounds tested were especially resistant. Dimethyl-substituted aliphatic acids and alcohols were also poorly utilized. Bacteria unable to grow on certain brominated fatty acids were capable of oxidizing and dehalogenating ω- but not α-bromoaliphatic acids.     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

Studies on the amino acid fermentation. Part 1. Production of L-glutamic acid by various microorganisms

Screening tests for glutamate producing strains were carried out, with the media containing carbohydrate and ammonia source as chief ingredients. Glutamate as well as certain other amino acids was detected by paper chromatography in culture broth of many microorganisms tested. Accumulation of L-glutamate in a significant amount (at least a few mg of glutamate per ml of broth) has been demonstrated by various strains of bacteria, streptomycetes, yeasts, and fungi. The highest level of glutamate production has been obtained by a new species of Micrococcus, yielding as much as 0.25 mole of it from one mole of glucose. The courses of fermentations mainly by known strains of microorganisms are shown. The importance of the cultural condition and strain specificity for the production of amino acids are briefly described.     

Hydrothermal production and characterization of protein and amino acids from silk waste

Non-catalytic hydrothermal decomposition of sericin and fibroin from silk waste into useful protein and amino acids was examined in a closed batch reactor at various temperatures, reaction times, and silk to water ratios to examine their effects on protein and amino acid yields. For the decomposition of sericin, the highest protein yield was found to be 0.466 mg protein/mg raw silk, obtained after 10 min hydrothermal reaction of silk waste at 1:100 silk to water ratio at 120 degrees C. The highest amino acid yield was found to be 0.203 mg amino acids/mg raw silk, obtained after 60 min of hydrothermal reaction of silk waste at 1:20 silk to water ratio at 160 degrees C. For the hydrothermal decomposition of fibroin, the highest protein yield was 0.455 mg protein/mg silk fibroin (1:100, 220 degrees C, 10 min) and that of amino acids was 0.755 mg amino acids/mg silk fibroin (1:50, 220 degrees C, 60 min). The rate of silk fibroin decomposition could be described by surface reaction kinetics. The soluble reaction products were freeze-dried to obtain sericin and fibroin particles, whose conformation and crystal structure of the particles were shown to differ from the original silk materials, particularly in the case of fibroin, in which the change from beta-sheet conformation to alpha-helix/random coil was observed.