A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Enhancement of erythritol production by Trichosporonoides oedocephalis ATCC 16958 through regulating key enzyme activity and the NADPH/NADP ratio with metal ion supplementation

Erythritol, a well-known natural sweetener, is mainly produced by microbial fermentation. Various metal ions (Al³⁺, Cu²⁺, Mn²⁺, and Ni²⁺) were added to the culture medium of Trichosporonoides oedocephalis ATCC 16958 at 30?mg/L in shake flask cultures. Compared with controls, Cu²⁺ increased the erythritol content by 86% and decreased the glycerol by-product by 31%. After 48 hr of shake flask culture, sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that expression levels of erythrose reductase (ER) in the presence of 30?mg/L CuSO₄?·?5H₂O were higher than those obtained after treatment with other examined metal ions. Furthermore, after 108 hr of batch culture in a 5-L bioreactor, supplementation with 30?mg/L of CuSO₄?·?5H₂O increased the specific erythritol content by 27%. Further studies demonstrated that ER activity under 30?mg/L CuSO₄?·?5H₂O supplementation in a fermentor was overtly increased compared with the control after 60 hr, while glycerol-3-phosphate dehydrogenase activity was clearly reduced in most of the fermentation process. Furthermore, the NADPH/NADP ratio was slightly lower in T. oedocephalis cells treated with Cu²⁺ compared with control cells. These results provide further insights into Cu²⁺ effects on erythritol biosynthesis in T. oedocephalis and should improve the industrial production of erythritol by biological processes.     

Recent advances in biological production of erythritol

Erythritol is a natural sweetener commonly used in the food and pharmaceutical industries. Produced by microorganisms as an osmoprotectant, it is an ideal sucrose substitute for diabetics or overweight persons due to its almost zero calorie content. Currently, erythritol is produced on an industrial scale through the fermentation of sugars by some yeasts, such as Moniliella sp. However, the popularity of erythritol as a sweetener is still small because of its high retail price. This creates an opportunity for further process improvement. Recent years have brought the rapid development of erythritol biosynthesis methods from the low-cost substrates, and a better understanding of the metabolic pathways leading to erythritol synthesis. The yeast Yarrowia lipolytica emerges as an organism effectively producing erythritol from pure or crude glycerol. Moreover, novel erythritol producing organisms and substrates may be taken into considerations due to metabolic engineering. This review focuses on the modification of erythritol production to use low-cost substrates and metabolic engineering of the microorganisms in order to improve yield and productivity.     

Screening and production of erythritol by newly isolated osmophilic yeast-like fungi

Twenty-eight erythritol-producing strains were isolated from pollen, honey and high sugar food samples collected in Taiwan. Amongst these, six strains (166-2, 262-1, 278-3, 440, 441 and 442) were high erythritol-producers with a yield higher than 30% for 30% glucose. The erythritol productivity of these strains ranged from 90.9 to 116.4 g l⁻¹. ¹H- and ¹³C-NMR analyses confirmed that the fermentation product was erythritol. The results of morphological and physiological studies indicate that strains 166-2, 262-1, 278-3, 440, and 442 may be members of the genus Moniliella. More studies are required to determine the taxonomic position of strain 441. The use of a medium containing 30% glucose and 1% yeast extract gave the highest erythritol productivity. On batch fermentation in a 5-l fermentor using strain 166-2, a maximal erythritol productivity of 111.0 g l⁻¹ was obtained after cultivation for 144 h.     

HOG1抑制剂调节球头三型孢菌多元醇生产及机理

目的:采用HOG1抑制剂对球头三型孢菌产多元醇进行调控。方法:向培养基中加入SB239063、SB202190和SB203580三种抑制剂进行发酵实验,比较三种抑制剂对发酵的影响。结果:实验结果表明SB239063可以降低球头三型孢菌细胞内胞浆3-磷酸甘油脱氢酶(ct GPD)的酶活,提高赤藓糖还原酶(ER)的酶活。此外对HOG1和Phospho-HOG1的Western blot结果分析显示,SB239063还会抑制球头三型孢菌细胞内HOG1的脱磷酸化。最终添加10μmol/L SB239063使发酵120 h后的多元醇产物中甘油产量下降20.57%,赤藓糖醇产量提高31.16%,底物转化率提高24.73%。结论:SB239063可以降低球头三型孢菌产甘油的能力,提高赤藓糖醇的产量。     

赤藓糖醇对主要致龋链球菌及耐氟菌株生长和产酸的影响

目的通过赤藓糖醇对变形链球菌、远缘链球菌及其耐氟菌株混合菌生长和产酸影响的体外研究,为赤藓糖醇防龋作用的机理提供制论依据。方法采用最小抑菌浓度递增法对变形链球菌（S.mutans ATCC 25175,S.m）、远缘链球菌（S.sobrinus 6715,S.s）进行氟化钠体外诱导耐氟菌株（S.m-FR、S.s-FR）,利用液体稀释法配制赤藓糖醇TSB液8个浓度,分别加入含有变形链球菌、远缘链球菌及其耐氟菌株的细菌混悬液48 h,用比浊法观察其对混合菌生长的影响,并用pH计测定培养前后上清液的△pH值。结果吸光度A值和△pH值实验前后与对照组相比最低浓度为12%时差异均有统计学意义（P〈0.05）,且随着浓度的升高A值和△pH值均下降。结论赤藓糖醇能抑制变形链球菌、远缘链球菌及耐氟菌株混合菌生长和产酸,并且随着浓度的升高抑制作用增强。     

Isolation of a novel high erythritol-producing <Emphasis Type="Italic">Pseudozyma tsukubaensis</Emphasis> and scale-up of erythritol fermentation to industrial level

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.     

Interactions between metal ions and carbohydrates. The coordination behavior of neutral erythritol to lanthanum and erbium ions

Lanthanide ions and erythritol form metal–alditol complexes with various structures. Lanthanum nitrate and erbium chloride coordinate to erythritol to give new coordination structures. The lanthanum nitrate–erythritol complex (LaEN), 2La(NO₃)₃·C₄H₁₀O₄·8H₂O, La³⁺ exhibits the coordination number of 11 (namely 11 polar atoms bound to one lanthanum) and is 11-coordinated to two hydroxyl groups from one erythritol molecule, six oxygen atoms from three nitrate ions and three water molecules. One erythritol molecule is coordinated to two La³⁺ ions and links the two metal ions together. The ratio of M:L is 2:1. The erbium chloride–erythritol complex (ErE), ErCl₂·C₄H₉O₄·2C₂H₅OH was obtained from ErCl₃ and erythritol in aqueous ethanol solution and the structure shows that deprotonation reaction occurs in the reaction process. The Er³⁺ cation is 8-coordinated with three hydroxyl groups of one erythritol molecule, two hydroxyl groups from another erythritol molecule, two ethanol molecules, and one chloride ion. Erythritol provides its three hydroxyl groups to one erbium cation and two hydroxyl groups to another erbium cation, that is, one hydroxyl group is coordinated to two metal ions and therefore loses its hydrogen atom and becomes a oxygen bridge. Another chloride ion is hydrogen bonded in the structure. The results indicate the complexity of metal–sugar coordination.     

Osmolytes and membrane lipids in the adaptation of micromycete Emericellopsis alkalina to ambient pH and sodium chloride

The accumulation of low molecular weight cytoprotective compounds (osmolytes) and changes in the membrane lipids composition are of key importance for the adaptation to stress impacts. However, the reason behind the wide variety of osmolytes present in the cell remains unclear. We suggest that specific functions of osmolytes can be revealed by studying the adaptation mechanisms of the mycelial fungus Emericellopsis alkalina (Hypocreales, Ascomycota) that is resistant to both alkaline pH values and high sodium chloride concentrations. It has been established that the fungus uses different osmolytes to adapt to ambient pH and NaCl concentration. Arabitol was predominant osmolyte in alkaline conditions, while mannitol prevailed in acidic conditions. On the salt-free medium mannitol was the main osmolyte; under optimal conditions (pH 10.2; 0.4 M NaCl) arabitol and mannitol were both predominant. Higher NaCl concentrations (1.0–1.5 M) resulted in the accumulation of low molecular weight polyol - erythritol, which amounted up to 12–14%, w/w. On the contrary, changes in the composition of membrane lipids were limited under pH and NaCl impacts; only higher NaCl concentrations led to the increase in the degree of unsaturation of membrane lipids. Results obtained indicated the key role of the osmolytes in the adaptation to the ambient pH and osmotic impacts.