Effect of sugars on D-arabitol production and glucose metabolism in Saccharomyces rouxii.

The effect of sugars on the production of d-arabitol and on the glucose catabolic pathways was investigated in the osmotrophic yeast Saccharomyces rouxii. The activity of d-arabitol dehydrogenase, which served as a measure of total d-arabitol production, increased when cells were grown in the presence of increasing glucose concentrations. Growth in sucrose had no effect on the enzyme activity. A high intracellular concentration of d-arabitol could be demonstrated when the cells were grown in a 60% glucose medium and could be eliminated by anaerobic growth or growth in the presence of 4 mg of chloramphenicol per ml. A mutant was isolated that would not grow in 60% glucose; although the regulation of d-arabitol dehydrogenase was altered in this strain, the production of d-arabitol was not eliminated. The activity of d-arabitol dehydrogenase followed the growth phases of the parent strain when the cells were preadapted to 30% glucose. If the cells were adapting from 1 to 30% glucose, a large increase in enzyme activity was detected before growth occurred. Protein synthesis was found to be involved in this increase in activity. There was an increased participation of the pentose phosphate pathway when the cells were grown in the presence of increasing glucose concentrations. The mutant strain had only an 11% pentose phosphate pathway participation compared with 20% for the parent strain in glucose. The results suggest that the active pentose phosphate pathway is involved in glucose tolerance by providing a plentiful supply of reduced nicotinamide adenine dinucleotide phosphate which is necessary for cell survival.     

D-Arabitol catabolic pathway in Klebsiella aerogenes

Klebsiella aerogenes strain W70 has an inducible pathway for the degradation of d-arabitol which is comparable to the one found in Aerobacter aerogenes strain PRL-R3. The pathway is also similar to the pathway of ribitol catabolism in that it is composed of a pentitol dehydrogenase, d-arabitol dehydrogenase (ADH), and a pentulokinase, d-xylulokinase (DXK). These two enzymes are coordinately controlled and induced in response to d-arabitol, the apparent inducer of synthesis of these enzymes. We obtained mutants which lacked a functional d-xylose pathway and were constitutive for the ribitol catabolic pathway. These mutants were able to grow on the unusual pentitol, xylitol, only if they contained the functional DXK of the d-arabitol pathway. This provided us with a specific selection technique for DXK(+) transductants. As in A. aerogenes, mutants constitutive for ADH were able to use this enzyme to convert the hexitol d-mannitol to d-fructose. With mutants blocked in the normal d-mannitol catabolic pathway, growth on d-mannitol became a test for ADH constitutivity. Growth of such mutants on xylitol, d-arabitol, and d-mannitol was utilized to classify transductants in mapping, by transductional analysis, the loci involved in d-arabitol utilization. Three-point crosses gave the order dalK-dalD-dalC, where dalK is the DXK structural gene, dalD is the ADH structural gene, and dalC is a regulatory site controlling synthesis of both enzymes.     

Molecular cloning and functional expression of d-arabitol dehydrogenase gene from Gluconobacter oxydans in Escherichia coli

A NADP-dependent d-arabitol dehydrogenase gene was cloned from Gluconobacter oxydans CGMCC 1.110 and functionally expressed in Escherichia coli. With d-arabitol as sole carbon source, E. coli transformants grew rapidly in minimal medium, and produced d-xylulose. The enzymatic properties of the 29kDa enzyme were documented. The DNA sequence surrounding the gene suggested that it is part of an operon with several components of a sugar alcohol transporter system, and the d-arabitol dehydrogenase gene belongs to the short-chain dehydrogenase family.     

Mutants of Aerobacter aerogenes capable of utilizing xylitol as a novel carbon

Wild-type Aerobacter aerogenes 1033 is unable to utilize xylitol. A succession of mutants was isolated capable of growth on this compound (0.2%) at progressively faster rates. Whereas the ability to utilize xylitol was achieved in the first-stage mutant (X1) by constitutive production of ribitol dehydrogenase (for which xylitol is a substrate but not an inducer), the basis for enhanced utilization of xylitol in the second-stage mutant (X2) was an alteration of ribitol dehydrogenase. This enzyme was purified from the various mutants. The apparent K(m) for xylitol was 0.12 m with X2 enzyme and 0.29 m with X1 enzyme. The X2 enzyme was also less heat stable and, at 0.05 m substrate concentration, had a higher ratio of activity with xylitol compared to ribitol than did the X1 enzyme. The third mutant (X3), with an even faster growth rate on xylitol, produced a ribitol dehydrogenase indistinguishable physically or kinetically from that of X2. However, X3 produced constitutively an active transport system which accepts xylitol. The usual function of this system is apparently for the transport of d-arabitol since the latter is not only a substrate but also an inducer of the transport system in parental strains of X3. The sequence of mutations described herein illustrates how genes belonging to different metabolic systems can be mobilized to serve a new biochemical pathway.     

Xylitol and D-arabitol toxicities due to derepressed fructose, galactitol, and sorbitol phosphotransferases of Escherichia coli.

d-Arabitol was observed to be toxic to many laboratory strains of Escherichia coli K-12, and xylitol was found to be toxic to an existing E. coli C mutant strain. Fructose-specific components of the phosphoenolpyruvate:sugar phosphotransferase system are required for xylitol toxicity. Selection for xylitol resistance results in Fru(-) strains blocked in fructose phosphotransferase. Introduction of the ptsF or ptsI mutation into a xylitol-sensitive strain eliminates sensitivity. [(14)C]fructose uptake experiments imply that the mutation to xylitol sensitivity, which is co-transducible with ara and leu, results in derepression of normally inducible fructose phosphotransferase. Wild-type strains also become xylitol sensitive if induced by (and then removed from) fructose. Xylitol toxicity is prevented by fructose in both wild-type and mutant strains. Circumstances causing xylitol, a new food additive, to become toxic to an otherwise insensitive wild-type organism have not been reported previously. The d-arabitol-sensitive laboratory strains are galactitol (dulcitol) utilizers, although most other strains are not. Selection for d-arabitol resistance results in Gat(-) strains blocked in a constitutive galactitol-specific component of the phosphotransferase system. A mutation causing d-arabitol sensitivity occurred many years ago in AB284, the parent of AB311, AB312, AB313, and many other strains. d-Arabitol sensitivity also occurs in sorbitol-constitutive strains and is shown, like the previous two instances of pentitol toxicities, to result from a constitutive phosphotransferase, which is blocked in mutants selected for resistance.     

Regulation of pentitol metabolism by Aerobacter aerogenes. I. Coordinate control of ribitol dehydrogenase and D-ribulokinase activities

Induction studies on Aerobacter aerogenes strain PRL-R3, using ribitol as the inducer-substrate, indicated that two enzymes of ribitol catabolism, ribitol dehydrogenase and d-ribulokinase, are coordinately induced. The utilization of d-arabinose as a substrate resulted in the induction of ribitol dehydrogenase as well as d-ribulokinase. Mutants which were constitutive for ribitol dehydrogenase were also constitutive for d-ribulokinase. In contrast, d-xylulokinase and d-arabitol dehydrogenase did not appear to be coordinately controlled. Induction studies and examination of d-arabitol dehydrogenase constitutive mutants indicated that the three enzymes of the converging pathways for d-arabitol and d-xylose catabolism are under separate control.     

Metabolism of some polyols by Rhizobium meliloti

The utilization of d-mannitol, d-arabitol, and d-sorbitol by Rhizobium meliloti was studied in extracts from mannitol-grown cells. Two different polyol dehydrogenases were induced by any of these polyols: (i) a nicotinamide adenine dinucleotide (NAD)-arabitol dehydrogenase and (ii) a NAD-sorbitol dehydrogenase, whereas polyol phosphate dehydrogenases were absent. d-Arabitol dehydrogenase was observed to act on both d-arabitol and d-mannitol, but d-sorbitol dehydrogenase acted specifically on d-sorbitol. d-Arabitol was oxidized to d-xylulose, d-mannitol and d-sorbitol were oxidized to d-fructose. An adenosine triphosphate-linked hexokinase which acts on d-fructose and absence of hexose isomerase were also detected in this organism.     

葡萄糖酸氧化杆菌木糖醇脱氢酶基因的克隆与表达

【目的】获得葡萄糖酸氧化杆菌(Gluconobacter oxydans CGMCC 1.637)的木糖醇脱氢酶基因,研究其酶学性质及碳源特别是D-阿拉伯醇和木糖醇对该酶活性的影响。【方法】通过已报道序列的木糖醇脱氢酶的保守区设计引物,用聚合酶链式反应(polymerase chain reaction,PCR)扩增获得目的基因片段。根据获得的片段序列设计引物克隆目的基因的5’和3’片段,将所获得的片段拼接,获得完整的木糖醇脱氢酶基因。通过构建工程菌获得重组蛋白,并利用氧化还原反应测定重组酶的活性。用含不同碳源的培养基培养G.oxydans CGMCC 1.637,并测定其破胞上清液木糖醇脱氢酶氧化木糖醇的活性;用不同碳源培养的G.oxydans CGMCC 1.637转化木酮糖,用高效液相色谱法测定木糖醇的产量。【结果】获得一个新的798bp的木糖醇脱氢酶基因,所编码的木糖醇脱氢酶含265个氨基酸,属于短链脱氢酶家族。酶学性质研究发现,该木糖醇脱氢酶催化木糖醇氧化的最适合条件为35℃、pH 10.0,最高活性为23.27 U/mg,催化木酮糖还原为木糖醇的最适条件为30℃、pH 6.0。最高活性为255.55 U/mg;该木糖醇脱氢酶的对木糖醇的Km和Vmax分别为78.97 mmol/L和40.17 U/mg。碳源诱导实验表明,d-山梨醇对G.oxydans CGMCC 1.637木糖醇脱氢酶的活性有明显的促进作用,而葡萄糖、果糖、木糖、木糖醇、D-阿拉伯醇对木糖醇脱氢酶活性有明显的抑制作用。而在转化实验中,用d-甘露糖培养的G.oxydans CGMCC 1.637的转化能力明显高于其他碳源培养的G.oxydans CGMCC 1.637的转化能力,其中,用阿拉伯醇培养的G.oxydans CGMCC 1.637的转化能力最低,仅为对照的35%。【结论】克隆自G.oxydans CGMCC 1.637的木糖醇脱氢酶基因是一个新的基因,用阿拉伯醇培养的G.oxydans CGMCC 1.637破胞液木糖醇脱氢酶活性低;且阿拉伯醇对G.oxydans CGMCC 1.637木酮糖的还原能力具有抑制作用。     

Effect of pentoses and pentitols on fermentation of hay by mixed populations of ruminal microorganisms

Consecutive batch culture, a technique which involves sequential transfer of cultures to fresh medium at regular intervals, was used to establish mixed ruminal-microbial populations in an anaerobic medium containing highly digestible hay. Once volatile fatty acid production was stable, perturbations were imposed in consecutive cultures by the addition of one of each of the following pentoses or analogous pentitols: l-arabinose, d-lyxose, d-ribose, d-xylose, l-arabitol, d-arabitol (lyxitol), ribitol, and xylitol. With the exception of d-lyxose, the addition of pentoses caused marked increases in propionate and valerate production, and except for d-arabitol, pentitol addition caused increases in butyrate and valerate production. On transfer to and continued incubation in the control medium, volatile fatty acid production reverted to preperturbed levels. The presence of pentitols and pentoses significantly reduced the endpoint pH of cultures and the proportion of hay that was fermented. With all added substrates, the response to the perturbation was at its maximum within one incubation (i.e., within 48 h). Similarly, the variables being monitored all returned to control levels within one incubation. On the basis of these results, it is suggested that changes were related to the need to maintain a redox balance within anaerobic cultures rather than any significant changes in the microbial population that was present.     

Production of d-arabitol from d-xylose by the oleaginous yeast Rhodosporidium toruloides IFO0880

Applied Microbiology and Biotechnology - The sugar alcohol d-arabitol is one of the Department of Energy’s top twelve bio-based building block chemicals. In this study, we found that the...     

Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans

Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8 Å. Gox2181 formed a homo-tetramer in the crystal that was coincident with the apparent molecular mass determined in the solution. Gox2181 displayed α/β-folding patterns, the conserved catalytic tetrad of Asn119-Ser147-Tyr162-Lys166, and the NAD-binding pocket, which aligned well with the ‘classical’ type of short-chain dehydrogenase/reductase (SDR) enzymes. Gox2181 was denoted SDR51C based on the SDR nomenclature system. The purified recombinant Gox2181 was demonstrated to be NAD(H)-dependent and active towards a wide range of substrates, including sugar alcohols, secondary alcohols, ketones, and ketoses. Among the substrates tested, Gox2181 displayed preference for secondary hydroxyl or carbonyl groups, showing low K_m values with d-arabitol and butanedione.     

Hydrolytic enzyme substrates. II. A new method for measuring periodate

This report describes an accurate and sensitive method for quantitatively measuring periodate concentration. The substances used to determine periodate are 4(p-nitrophenoxy)1,2-butanediol and 4(2,4-dinitrophenoxy)1,2-butanediol. These substances are readily oxidized by periodate yielding β-nitrophenoxy aldehydes which undergoes a facile β-elimination in base to yield the colored nitrophenolate ion. The concentration of the nitrophenolate ion is thus equivalent to the concentration of periodate. This report documents the validity of this reaction as an analytical method. The method was shown to be capable of accurately measuring periodate in concentrations as low as 10^?8M. Its value in biochemical analyses was demonstrated by quantitatively measuring the amount of periodate used to oxidize small quantities of adenosine 5′-phosphate, d-arabitol and d-glucose. Its accuracy, sensitivity and ease of use was shown by its utility in estimating the molecular weight of yeast transfer RNA using about 6 A₂₆₀ units of this material.     

Formation of glucose from hexoses, pentoses, polyols and related substances in kidney cortex

1. Slices of rat kidney cortex, on incubation in a saline medium, formed d-glucose from the following substances: d-fructose, d-galactose, d-mannose, l-sorbose, l-arabinose, d-xylose, glycerol, myo-inositol, l-iditol, sorbitol, xylitol, ribitol, methylglyoxal, dihydroxyacetone, l-glyceraldehyde, d-glyceraldehyde, dl-glyceraldehyde, dl-glycerate. Values for the rates of glucose formation from these precursors are given. 2. No glucose was formed from l-rhamnose, d-arabitol, d-arabinose, d-ribose, l-fucose, d-lyxose, mannitol, dulcitol, d-glucuronate, propane-1,2-diol and propan-2-ol. 3. The pathways of glucose formation from the various precursors are discussed (Scheme 1). 4. l-Glyceraldehyde inhibited the formation of glucose from d-glyceraldehyde.     

Polyol permeability of the human red cell. Interpretation of glucose transport in terms of a pore

The kinetic equations describing transport through a pore that has a binding site and that undergoes a conformational change are identical to those of a carrier model. Therefore, in order to distinguish between the two models it is necessary to test specific predictions based on detailed mechanistic models. A pore model is described in which the substrate (glucose) is able to reach the single binding site only from the outside when the pore is in conformation I and only from the inside when it is conformation II. On the basis of this model it is predicted that solutes which do not have any specific affinity for the binding site should still have a finite permeability via the glucose transport system if they are the same size or smaller than glucose. This permeability should be proportional to the volume of distribution of the solute in the pore and should therefore decrease with increasing molecular size. A geometric pore volume can be estimated from this size dependence. In order to test these predictions, the glucose-dependent permeability of a series of 4-carbon (erythritol), 5-carbon (d-arabitol, l-arabitol and xylitol) and 6-carbon (d-mannitol, d-sorbitol and $\text{myo-}\text{inositol}$) polyols was measured. The permeability of all the polyols is decreased by the presence of glucose and the $\text{K}{}_{\mathrm{<mtext>I</mtext>}}$ of this “inhibitable” component is similar to that of d-sorbose, suggesting that this component is associated with the glucose transport system. Since these observations could be explained entirely in terms of a specific affinity for a carrier binding site, they do not exclude a carrier mechanism. However, as predicted for the pore model, this “inhibitable” permeability decreased with increasing molecular size and the calculated geometric pore volume was of a size that would be expected for a cell membrane pore.     

A transfer-function representation for regulatory responses of a controlled metabolic pathway

A transfer-function representation for the response of a controlled metabolic pathway to the changes in influx and efflux rates of metabolites is formulated to describe analytically and approximately the regulatory behavior of the pathway around a steady state. The pathway model analysed is an open and homogeneous system which consists of two consecutive enzymatic reactions catalyzed by an allosteric enzyme of Monod-Wyman-Changeux (MWC) dimeric model and a Michaelis-Menten-type enzyme, respectively, and undergoes the feedback inhibition by the end product. The rate equation for the system (a system of ordinary differential equations) is linearized about a steady state, so that the responses of the reaction rates to the changes in influx rate of the substrate and efflux rate of the end product are expressed in a form of transfer function. The formulation leads to the transfer function for the response of production rate of the end product to the change in its efflux rate to clarify the regulatory response of feedback mechanism in controlled metabolic pathways. The relationship among the chemical species in the system at steady stete also supports a reasonable assumption that the regulatory mechanisms in metabolic pathways are to control the production of end product against the change in its demand from the cellular environments.     

cGAS-STING信号通路在畜禽疾病中的研究进展

cGAS-STING信号通路是一种细胞内DNA感受器,可以识别自身病变或外部进入细胞质中的双链DNA。其不仅与肿瘤、病毒和细菌感染及自身免疫系统疾病密切相关,而且在非特异性免疫系统中发挥重要作用。截至目前,国内外对于cGAS-STING信号通路的研究主要集中在哺乳动物肿瘤相关性疾病及与先天性免疫系统相关的疾病中,而通路对畜禽疾病调控的影响机制非常重要。本文以cGAS-STING信号通路在宿主受到病原感染中发挥的作用为切入点,对其在不同畜禽病原感染中的调控作用进行综合论述,以期为畜禽疾病的防控提供理论依据和参考。     

The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation

This review outlines the mechanisms underlying the interaction between the nervous and immune systems of the host in response to an immune challenge. The main focus is the cholinergic anti-inflammatory pathway, which we recently described as a novel function of the efferent vagus nerve. This pathway plays a critical role in controlling the inflammatory response through interaction with peripheral a7 subunit-containing nicotinic acetylcholine receptors expressed on macrophages. We describe the modulation of systemic and local inflammation by the cholinergic anti-inflammatory pathway and its function as an interface between the brain and the immune system. The clinical implications of this novel mechanism also are discussed.     

C3a is required for the production of CXC chemokines by tubular epithelial cells after renal ishemia/reperfusion

The complement system is one of the major ways by which the body detects injury to self cells, and the alternative pathway of complement is rapidly activated within the tubulointerstitium after renal ischemia/reperfusion (I/R). In the current study, we investigate the hypothesis that recognition of tubular injury by the complement system is a major mechanism by which the systemic inflammatory response is initiated. Gene array analysis of mouse kidney following I/R initially identified MIP-2 (CXCL2) and keratinocyte-derived chemokine (KC or CXCL1) as factors that are produced in a complement-dependent fashion. Using in situ hybridization, we next demonstrated that these factors are expressed in tubular epithelial cells of postischemic kidneys. Mouse proximal tubular epithelial cells (PTECs) in culture were then exposed to an intact alternative pathway and were found to rapidly produce both chemokines. Selective antagonism of the C3a receptor significantly attenuated production of MIP-2 and KC by PTECs, whereas C5a receptor antagonism and prevention of membrane attack complex (MAC) formation did not have a significant effect. Treatment of PTECs with an NF-kappaB inhibitor also prevented full expression of these factors in response to an intact alternative pathway. In summary, alternative pathway activation after renal I/R induces production of MIP-2 and KC by PTECs. This innate immune system thereby recognizes hypoxic injury and triggers a systemic inflammatory response through the generation of C3a and subsequent activation of the NF-kappaB system.     

D-apiose reductase from Aerobacter aerogenes

A strain of Aerobacter aerogenes PRL-R3 has been isolated which utilizes d-apiose as its sole source of carbon. A new enzyme, d-apiose reductase, was discovered in this strain. The enzyme was not present when the strain was grown on d-glucose. d-Apiose reductase catalyzes the nicotinamide adenine dinucleotide-dependent interconversion of d-apiose and d-apiitol. The enzyme is specific for d-apiose and d-apiitol, with a few possible exceptions. The K(m) for d-apiose is 0.02 m. The K(m) for d-apiitol is 0.01 m. The enzyme is almost completely specific for the reduced and oxidized forms of nicotinamide adenine dinucleotide. When cell-free extracts were centrifuged at 100,000 x g for 1 hr, the enzyme remained in solution. Optimal activity for the reduction of d-apiose was obtained at pH 7.5 in glycylglycine buffer, whereas for the oxidation of d-apiitol it was obtained at pH 10.5 in glycine buffer. Enzymatic reduction of d-apiose was not appreciably affected by the presence of 0.02 m ethylenediaminetetraacetate. Paper chromatography and specific spray reagents were used to identify d-apiitol and d-apiose as the products of this reversible reaction. d-Apiose and d-apiitol did not serve as substrates for ribitol dehydrogenase and d-arabitol dehydrogenase from A. aerogenes PRL-R3.