Fluoride‐sensitivity of growth and acid production of oral Actinomyces: comparison with oral Streptococcus

Actinomyces are predominant oral bacteria; however, their cariogenic potential in terms of acid production and fluoride sensitivity has not been elucidated in detail and compared with that of other caries‐associated oral bacteria, such as Streptococcus. Therefore, this study aimed to elucidate and compare the acid production and growth of Actinomyces and Streptococcus in the presence of bicarbonate and fluoride to mimic conditions in the oral cavity. Acid production from glucose was measured by pH‐stat at pH 5.5 and 7.0 under anaerobic conditions. Growth rate was assessed by optical density in anaerobic culture. Although Actinomyces produced acid at a lower rate than did Streptococcus, their acid production was more tolerant of fluoride (ID_{acid production} 50 = 110–170 ppm at pH 7.0 and 10–13 ppm at pH 5.5) than that of Streptococcus (ID_{acid production} 50 = 36–53 ppm at pH 7.0 and 6.3–6.5 ppm at pH 5.5). Bicarbonate increased acid production by Actinomyces with prominent succinate production and enhanced their fluoride tolerance (ID_{acid production} 50 = 220–320 ppm at pH 7.0 and 33–52 ppm at pH 5.5). Bicarbonate had no effect on these variables in Streptococcus. In addition, although the growth rate of Actinomyces was lower than that of Streptococcus, Actinomyces growth was more tolerant of fluoride (ID_growth 50 = 130–160 ppm) than was that of Streptococcus (ID_growth 50 = 27–36 ppm). These results indicate that oral Actinomyces are more tolerant of fluoride than oral Streptococcus, and bicarbonate enhances the fluoride tolerance of oral Actinomyces. Because of the limited number of species tested here, further study is needed to generalize these findings to the genus level.     

Metabolic origin of δ15N values in nitrogenous compounds from Brassica napus L. leaves

Nitrogen isotope composition (δ¹⁵N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ¹⁵N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ¹⁵N. Here, we investigated the δ¹⁵N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ¹⁵N between nitrogenous compounds (up to 30‰) and the content in (¹⁵N enriched) nitrate had a clear influence on leaf δ¹⁵N. Using a simple steady‐state model of day metabolism, we suggest that the δ¹⁵N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme‐catalysed reactions in primary nitrogen metabolism. δ¹⁵N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ¹⁵N value of leaf nitrogenous compounds.     

Optimization of medium components for high-molecular-weight hyaluronic acid production by <Emphasis Type="Italic">Streptococcus</Emphasis> sp. ID9102 via a statistical approach

Hyaluronic acid (HA), linear high-molecular-weight glycosaminoglycan produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. In this paper, we report on the optimization of medium components for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) by two-step optimization (one-factor-at-a-time and taguchi orthogonal array design). In the first step, medium components, such as carbon, nitrogen, phosphate, and mineral sources, were selected for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) using the one-factor-at-a-time method. In the second step, the concentration of the selected medium components was optimized using taguchi orthogonal array design. The design for medium optimization was developed and analyzed using MINITAB 14 software. In addition, the effect of amino acid and organic acid, such as glutamine, glutamate, and oxalic acid, was studied for HA production in Streptococcus sp. ID9102 (KCTC 11935BP). Through these processes, the optimum medium comprising 4% glucose, 0.75% yeast extract, 1.0% casein peptone, 0.25% K₂HPO₄, 0.05% MgCl₂, 0.5% NaCl, 0.04% glutamine, 0.06% glutamate, and 0.02% oxalic acid was determined. We were able to produce HA with a molecular weight of 5.9 × 10⁶ at a productivity of 6.94 g/l on pilot scale fermentation.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

The Production of Organic Acids

Abstract

The production of organic acids covers two aspects: first, the metabolic pathways involved in the biosynthesis, and, second, the industrial process strategy adopted. The review seeks to show the underlying biochemical similarities in the biosynthesis of organic acids and the resulting similarities in the commercial processes.

Two groups of acids are defined, those with a “long” biosynthetic path from glucose, involving much of the glycolytic pathway and the tricarboxylic acid cycle, and those acids with a “short pathway”, essentially a biotransformation of glucose. The regulation of the pathways and the future developments in metabolic control theory and genetic manipulations relating to them are considered. The organisms used industrially are also limited, Aspergillus sp. and Candida yeasts; again the underlying metabolic similarities lead to similar strategies for all the acids discussed.     

Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated protein

Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully ¹³C‐labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Metabolic costs of amino acid and protein production in Escherichia coli

Escherichia coli is the most popular microorganism for the production of recombinant proteins and is gaining increasing importance for the production of low-molecular weight compounds such as amino acids. The metabolic cost associated with the production of amino acids and (recombinant) proteins from glucose, glycerol and acetate was determined using three different computational techniques to identify those amino acids that put the highest burden on the biosynthetic machinery of E. coli. Comparing the costs of individual amino acids, we find that methionine is the most expensive amino acid in terms of consumed mol of ATP per molecule produced, while leucine is the most expensive amino acid when taking into account the cellular abundances of amino acids. Moreover, we show that the biosynthesis of a large number of amino acids from glucose and particularly from glycerol provides a surplus of energy, which can be used to balance the high energetic cost of amino acid polymerization.     

Catabolic pathways of glucose in Bacillus circulans var. alkalophilus

Enzymes and the metabolic pathways of glucose catabolism of Bacillus circulans var. alkalophilus were studied. The metabolism of the microbe was mixed acid fermentative yielding mainly acetic and formic acids as end products from glucose. It was estimated that B. circulans var. alkalophilus partitions 90%–93% of the carbon from glucose into the Embden-Meyerhof-Parnas (EMP) pathway and 7%–10% into the hexose monophosphate (HMP) and Entner-Doudoroff (ED) pathways. Rather low activities of glucose dehydrogenase and gluconokinase appeared in the early logarithmic and late stationary phases, whereas NADH oxidase was markedly high. This result can be explained by a demand to reduce NADH to NAD⁺ for the EMP pathway; when acetic and formic acids are produced, no NADH is regenerated to NAD⁺, which is required in the early steps of EMP and HMP pathways. A small percentage (1.6%–2.4%) of the total CO₂ was formed from (6-C) of glucose, which means that the tricarboxylic acid cycle was functional but its contribution was insignificant. Large differences do not seem to exist between alkaliphilic and neutrophilic bacilli in the use of glucose pathways. Received: January 29, 1999 / Accepted: July 30, 1999     

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers

Polylactic acid (PLA) is a promising biomass‐derived polymer, but is currently synthesized by a two‐step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), by direct fermentation of metabolically engineered Escherichia coli. As shown in an accompanying paper, introduction of the heterologous metabolic pathways involving engineered propionate CoA‐transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl‐CoA and incorporation of lactyl‐CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB‐co‐LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome‐scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB‐co‐LA) copolymers containing 55–86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB‐co‐LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio‐based one‐step production of PLA and its copolymers. This strategy should be generally useful for developing other engineered organisms capable of producing various unnatural polymers by direct fermentation from renewable resources. Biotechnol. Bioeng. 2010; 105: 161–171. © 2009 Wiley Periodicals, Inc.     

The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control

The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonine's contribution being ～ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end‐products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7‐fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8‐fold reduction in threonine‐derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.     

感受态与口腔链球菌多种表型间关系的研究进展

链球菌是人类口腔中最为常见的细菌类群之一,在口腔微生态平衡的维持与致病中发挥了重要作用。口腔链球菌中的大多数可以进入感受态,在此生理状态下,细菌可摄取环境中的DNA并整合进入自身基因组从而获得新的遗传表型或特性。大量研究表明,口腔链球菌的感受态调控通路不是孤立的,与生物膜形成、细菌素产生、耐酸、氧应激、细胞自溶和耐药性等多个表型的调控存在紧密关系,研究这些不同表型间的相互影响对理解口腔菌群稳态及防治疾病有重要意义。本文以变异链球菌、格氏链球菌、血链球菌和肺炎链球菌4种典型的口腔链球菌为代表,对感受态与口腔链球菌多种表型间关系的研究进展做一综述。     

Enhanced Biosynthesis of Sulfide Oxidase by Arthrobacter Species Using Response Surface Methodology

Response surface methodology was used to develop a fermentation medium for the enhanced biosynthesis of a novel sulfide oxidase by Arthrobacter species strain FR‐3. The interactive effect of the medium components – such as glucose as the carbon source, and tryptone and yeast extract as the nitrogen source – was evaluated by a 2³‐factorial central composite statistical design. Glucose and yeast extract were found to be the more influential medium constituents compared to tryptone since they had lower coefficients of linear effect, P‐values (< 0.02). The optimal fermentation medium components for the enhanced production of sulfide oxidase were recorded as glucose (8.98 g/L), tryptone (10.62 g/L) and yeast extract (7.3 g/L). Optimization of the medium constituents increased the experimental enzyme yield by 54 % compared to the unoptimized medium. This is the first report on the overproduction of sulfide oxidase by using response surface methodology.