Enhancing thermostability of -mannanase by protective additives

The effects of some sugars (glucose, mannose, fructose, sucrose and chitosan) and polyols (glycol, glycerol and sorbitol) as protective additive on the thermostability of ?-mannanase were studied. The optimal reaction temperatures of ?-mannanase and the thermodynamics and the deactivation kinetics with or without additives were also investigated. The experimental results show that sucrose, chitosan and sorbitol could apparently improve the thermal stability of ?-mannanase when their concentration was kept at 2 g/L. The optimal combination additive proportion was sucrose: chitosan : sorbitol = 1 : 2 : 2 (molar ratio) using the orthogonal experimental design. The sucrose, chitosan, glycerol, sorbitol and the combination additive might increase the optimal reaction temperature from 50°C to about 60°C due to their good protection effect. The thermal deactivation curves of ?-mannanase accorded with the kinetic rules of first order reaction, and the corresponding kinetic and thermodynamic parameters were calculated. Meanwhile, the protective mechanism of the additives against deactivation of enzyme was also discussed.     

Effects of three main sugars in cane molasses on the production of butyric acid with <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis>

The effects of three main sugars in cane molasses were investigated systematically to prepare a cost-effective medium for butyric acid bioproduction. Additionally, 30 g/L corn steep liquor was screened out as the suitable nitrogen source. In the batch fermentation of free cells, when 60 g/L glucose was the only carbon source, 21.28 g/L butyric acid was achieved after 30 h cultivation. Similar product concentration, productivity and yield were obtained when 60 g/L fructose was applied. The utilization of sucrose would bring about lower productivity (0.29 g/L·h) and product concentration (18.15 g/L), but the yield of butyric acid/sucrose (0.34 g/g) is almost the same as that from glucose or fructose (0.35 g/g). Finally, the sugar mixture (15 g/L glucose, 20 g/L fructose and 35 g/L sucrose) was employed to produce butyric acid in a fibrous-bed bioreactor (FBB), and 40.11 g/L butyric acid was produced with one simple fed-batch strategy.     

Sugar Transporters in Plasmodiophora brassicae: Genome-Wide Identification and Functional Verification

Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host’s carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host.     

碳源对土曲霉中洛伐他汀生物合成的影响

通过摇瓶培养考察碳源种类及其初始水平对土曲霉次级代谢物洛伐他汀生物合成的影响。蔗糖和葡萄糖等快速利用碳源产洛伐他汀量较低,且易积累其直接前体monacolin J;淀粉和甘油等缓慢利用碳源有利于细胞生长和产物合成,其monacolin J积累相对较少,甘油为碳源时,可获得最高产物量(937.5±12.3)mg/L,乳糖不利于细胞生长导致较低的产物量。以淀粉为碳源时,其初始水平的增加可促进洛伐他汀的合成,碳质量分数为2%时对底物利用率较高,4%时最高产物量可达(875.4±23.1)mg/L,monacolin J则在2%—3%时积累较多,同时产生较多的2-甲基丁酸。     

Enhancing thermostability of β-mannanase by protective additives

The effects of some sugars (glucose, mannose, fructose, sucrose and chitosan) and polyols (glycol, glycerol and sorbitol) as protective additive on the thermostability of β-mannanase were studied. The optimal reaction temperatures of β-mannanase and the thermodynamics and the deactivation kinetics with or without additives were also investigated. The experimental results show that sucrose, chitosan and sorbitol could apparently improve the thermal stability of β-mannanase when their concentration was kept at 2 g/L. The optimal combination additive proportion was sucrose: chitosan : sorbitol = 1 : 2 : 2 (molar ratio) using the orthogonal experimental design. The sucrose, chitosan, glycerol, sorbitol and the combination additive might increase the optimal reaction temperature from 50°C to about 60°C due to their good protection effect. The thermal deactivationcurves of β-mannanase accorded with the kinetic rules of first order reaction, and the corresponding kinetic and thermodynamic parameters were calculated. Meanwhile, the protective mechanism of the additives against deactivation of enzyme was also discussed. __________ Translated from Journal of Tianjin University, 2007, 41(1): 114–118 [译自: 天津大学学报]     

Biochemistry of Citric Acid Production from Rapeseed Oil by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Yeast

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica NG40/UV7 as well as the biosynthesis of citric and isocitric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is its hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites were changed insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids available in the medium do not suppress the metabolism of each other. Citric acid production and a ratio between citric and isocitric acids depended on the strain used. It was revealed that the wild strain produced almost equal amounts of citric and isocitric acids while the mutant produced only citric acid (175 g/L) with a yield of 1.5 g of CA per g of oil.     

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol–oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO₃, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 μg/g of kynurenic acid, which increases the health-promoting value of the product.     

Adult Parasitoids of Honeydew-Producing Insects Prefer Honeydew Sugars to Cover their Energetic Needs

To meet their carbohydrate requirements, adult parasitoids exploit a broad range of sugar resources, including floral and extrafloral nectar and honeydew. Although honeydew might be the predominant sugar source, especially in agricultural systems, it often is nutritionally inferior to sugar sources like nectar. Given its broad availability, it may be expected that sugar-feeding insects have evolved specialized adaptations to deal with this typically inferior sugar source. This would apply especially to organisms that have a close association with honeydew producers. Here, we hypothesized that parasitoids of honeydew-producing insects should show a pronounced response to sugars, such as fructose, sucrose, melezitose, and trehalose, and to a lesser extent glucose. To test this hypothesis, we investigated sugar consumption, feeding behavior and survival of the aphid parasitoid Aphidius ervi on several sugars (equiweight solutions). Our results show that A. ervi adults consumed typical honeydew sugars (sucrose, fructose, trehalose, and melezitose) the most, while consuming considerably less glucose or melibiose. Rhamnose, which does not occur in aphid honeydew, was not, or was only marginally, consumed. When different sugars were provided at the same time, A. ervi adults preferred sucrose or fructose over glucose or melezitose. Furthermore, pre-exposure to sucrose or fructose significantly reduced subsequent intake of glucose, suggesting an acquired distaste for glucose after being previously exposed to highly preferred sugars such as sucrose and fructose. Altogether, this study shows that A. ervi adults prefer sugars (fructose, melezitose, trehalose, and sucrose) that are overrepresented in aphid honeydew and show a lower preference to one (glucose) that is underrepresented in honeydew.     

CsSWEET2, a Hexose Transporter from Cucumber (Cucumis sativus L.), Affects Sugar Metabolism and Improves Cold Tolerance in Arabidopsis

Sugars, which are critical osmotic compounds and signalling molecules in plants, and Sugars Will Eventually be Exported Transporters (SWEETs), which constitute a novel family of sugar transporters, play central roles in plant responses to multiple abiotic stresses. In the present study, a member of the SWEET gene family from cucumber (Cucumis sativus L.), CsSWEET2, was identified and characterized. Histochemical analysis of β-glucuronidase expression in transgenic Arabidopsis plants showed that CsSWEET2 is highly expressed in the leaves; subcellular localization indicated that CsSWEET2 proteins are localized in the plasma membrane and endoplasmic reticulum. Heterologous expression assays in yeast demonstrated that CsSWEET2 encodes an energy-independent hexose/H⁺ uniporter that can complement both glucose and fructose transport deficiencies. Compared with wild-type Arabidopsis plants, transgenic Arabidopsis plants overexpressing CsSWEET2 had much lower relative electrolyte leakage levels and were much more resistant to cold stress. Sugar content analysis showed that glucose and fructose levels in the transgenic Arabidopsis plants were significantly higher than those in the wild-type plants. Taken together, our results suggest that, by mediating sugar metabolism and compartmentation, CsSWEET2 plays a vital role in improving plant cold tolerance.     

基于果糖与葡萄糖不同混合比例的丙酮丁醇发酵

介绍了以不同底物的丁醇发酵结果,阐述了在以55g/L葡萄糖与果糖（1∶4）混合糖模拟菊芋物料为底物的丁醇发酵过程中存在果糖利用及丁醇产量较低等问题,研究了基于葡萄糖与果糖不同混合比例（1∶2、2∶3、3∶2及3∶1）的丁醇发酵性能。研究结果说明了随着混合比例提高,发酵时间由76h缩短至48h,菌体最大生物量OD620由2.1提高至4.3,而当葡萄糖与果糖混合比例为1∶2时,发酵过程中菌体细胞对果糖代谢能力最佳,且终点残糖浓度仅为2.1g/L,果糖利用效率达到95.03%,丁醇及总溶剂产量分别达到9.7g/L与16.0g/L。     

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.     

A Novel Hexose Transporter ChHxt6 Is Required for Hexose Uptake and Virulence in Colletotrichum higginsianum

Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose.     

Effects of Different Oil Sources and Residues on Biomass and Metabolite Production by Yarrowia lipolytica YB 423-12

Yarrowia lipolytica is known to have the ability to assimilate hydrophobic substrates like triglycerides, fats, and oils, and to produce single-cell oils, lipases, and organic acids. The aim of the present study was to investigate the effects of different oil sources (borage, canola, sesame, Echium, and trout oils) and oil industry residues (olive pomace oil, hazelnut oil press cake, and sunflower seed oil cake) on the growth, lipid accumulation, and lipase and citric acid production by Y. lipolytica YB 423-12. The maximum biomass and lipid accumulation were observed with linseed oil. Among the tested oil sources and oil industry residues, hazelnut oil press cake was the best medium for lipase production. The Y. lipolytica YB 423-12 strain produced 12.32 ± 1.54 U/mL (lipase activity) of lipase on hazelnut oil press cake medium supplemented with glucose. The best substrate for citric acid production was found to be borage oil, with an output of 5.34 ± 0.94 g/L. The biotechnological production of valuable metabolites such as single-cell oil, lipase, and citric acid could be achieved by using these wastes and low-cost substrates with this strain. Furthermore, the cost of the bio-process could also be significantly reduced by the utilization of various low-cost raw materials, residues, wastes, and renewable resources as substrates for this yeast.     

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.     

Bacterial Cellulose Production from Industrial Waste and by-Product Streams

The utilization of fermentation media derived from waste and by-product streams from biodiesel and confectionery industries could lead to highly efficient production of bacterial cellulose. Batch fermentations with the bacterial strain Komagataeibacter sucrofermentans DSM (Deutsche Sammlung von Mikroorganismen) 15973 were initially carried out in synthetic media using commercial sugars and crude glycerol. The highest bacterial cellulose concentration was achieved when crude glycerol (3.2 g/L) and commercial sucrose (4.9 g/L) were used. The combination of crude glycerol and sunflower meal hydrolysates as the sole fermentation media resulted in bacterial cellulose production of 13.3 g/L. Similar results (13 g/L) were obtained when flour-rich hydrolysates produced from confectionery industry waste streams were used. The properties of bacterial celluloses developed when different fermentation media were used showed water holding capacities of 102–138 g·water/g·dry bacterial cellulose, viscosities of 4.7–9.3 dL/g, degree of polymerization of 1889.1–2672.8, stress at break of 72.3–139.5 MPa and Young’s modulus of 0.97–1.64 GPa. This study demonstrated that by-product streams from the biodiesel industry and waste streams from confectionery industries could be used as the sole sources of nutrients for the production of bacterial cellulose with similar properties as those produced with commercial sources of nutrients.     

Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.     

A Comparative Genomic and Safety Assessment of Six Lactiplantibacillus plantarum subsp. argentoratensis Strains Isolated from Spontaneously Fermented Greek Wheat Sourdoughs for Potential Biotechnological Application

The comparative genome analysis of six Lactiplantibacillus plantarum subsp. argentoratensis strains previously isolated from spontaneously fermented Greek wheat sourdoughs is presented. Genomic attributes related to food safety have been studied according to the European Food Safety Authority (EFSA) suggestions for the use of lactic acid bacteria (LAB) in the production of foods. Bioinformatic analysis revealed a complete set of genes for maltose, sucrose, glucose, and fructose fermentation; conversion of fructose to mannitol; folate and riboflavin biosynthesis; acetoin production; conversion of citrate to oxaloacetate; and the ability to produce antimicrobial compounds (plantaricins). Pathogenic factors were absent but some antibiotic resistance genes were detected. CRISPR and cas genes were present as well as various mobile genetic elements (MGEs) such as plasmids, prophages, and insertion sequences. The production of biogenic amines by these strains was not possible due to the absence of key genes in their genome except lysine decarboxylase associated with cadaverine; however, potential degradation of these substances was identified due to the presence of a blue copper oxidase precursor and a multicopper oxidase protein family. Finally, comparative genomics and pan-genome analysis showed genetic differences between the strains (e.g., variable pln locus), and it facilitated the identification of various phenotypic and probiotic-related properties.     

固相萃取和高效液相色谱法测定水果中的糖

研究了高效液相色谱法测定水果中的糖。水果样品的糖提取液用WatersSep Pak C1 8固相萃取小柱预分离 ,以WatersSugar Pak- 1 钙型阳离子交换柱为固定相 ,0 .0 5 g·L- 1 EDTA钙钠水溶液为流动相分离 ,示差折光仪为检测器检测。蔗糖、葡萄糖和果糖的检测限分别为 2mg/L、4mg/L和 4mg/L ,相对标准偏差在 0 .76%～1.8%之间 ,标准回收率在 97%～ 10 3 %之间。方法用于几种水果样品的测定 ,结果满意。     

Feeding responses of eastern spruce budworm larvae to sucrose and other carbohydrates

Spruce budworm larvae reared on an artificial diet were tested for preference of twelve carbohydrates over a water control. A strong preference for sucrose was seen, followed by fructose, inositol, and glucose. Male and female larvae do not differ in their responses to sucrose. The behavioral threshold for sucrose is at 10^–4 to 10^–3 M, with a peak response in the range of 0.01 to 0.05 M sucrose. Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae).Work leading to this publication was funded in part by a USDA Forest Service sponsored program entitled Canada/United States Spruce Budworms Program (grant 23–352), and in part by the National Sciences and Engineering Council of Canada (operating grant A9723), and in part by the Whitehall Foundation.