Trehalose synthases from the subfamily GH13_16 involved in α-glucan biosynthesis - a focus on their maltokinase domain.

The subfamily GH13_16 trehalose synthase (TreS) converts maltose to trehalose and vice versa. Typically, it consists of three domains, but it may contain a C-terminal extension exhibiting clear sequence features of a maltokinase (MaK). The present in silico study was focused on collection of naturally fused TreS-MaKs and their subsequent detailed bioinformatics analysis. Hence a set of total 3354 unique sequences was compared consisting of 1900 single TreSs, 1426 fused TreS-MaKs and 28 single MaKs. Fused TreS-MaKs were divided into five groups, namely with a standard MaK, with mutations in the maltose-binding site, of the catalytic nucleophile, of the general acid/base and of both catalytic residues. Sequence logos bearing the best conserved sequence regions were prepared for both TreSs and MaKs in an effort to find unique sequence features. In addition, linkers connecting the TreS and MaK parts in the fused enzymes were analysed. This analysis revealed that MaKs in fused enzymes have an extended N-terminal regions compared to single MaKs. Finally, the evolutionary relationships were demonstrated by phylogenetic trees of TreS parts from single TreSs and fused TreS-MaKs from the same organism as well as of single TreSs existing in multiple isoforms in the same organism.

NsdD, a GATA-type transcription factor is involved in regulation and biosynthesis of macromolecules melanin, pullulan, and polymalate in Aureobasidium melanogenum.

In this study, an NSDD gene, which encoded a GATA-type transcription factor involved in the regulation and biosynthesis of melanin, pullulan, and polymalate (PMA) in Aureobasidium melanogenum, was characterized. After the NSDD gene was completely removed, melanin production by the Δnsd mutants was enhanced, while pullulan and polymalate production was significantly reduced. Transcription levels of the genes involved in melanin biosynthesis were up-regulated while expression levels of the genes responsible for pullulan and PMA biosynthesis were down-regulated in the Δnsdd mutants. In contrast, the complementation of the NSDD gene in the Δnsdd mutants made the overexpressing mutants restore melanin production and transcription levels of the genes responsible for melanin biosynthesis. Inversely, the complementation strains, compared to the wild type strains, showed enhanced pullulan and PMA yields. These results demonstrated that the NsdD was not only a negative regulator for melanin biosynthesis, but also a key positive regulator for pullulan and PMA biosynthesis in A. melanogenum. It was proposed how the same transcriptional factor could play a negative role in melanin biosynthesis and a positive role in pullulan and PMA biosynthesis. This study provided novel insights into the regulatory mechanisms of multiple A. melanogenum metabolites and the possibility for improving its yields of some industrial products through genetic approaches.

Effect of highly branched α-glucans synthesized by dual glycosyltransferases on the glucose release rate.

Increasing α-1,6 linkages in starch molecules generates a large amount of α-limit dextrins (α-LDx) during α-amylolysis, which decelerate the release of glucose at the intestinal α-glucosidase level. This study synthesized highly branched α-glucans from sucrose using Neisseria polysaccharea amylosucrase and Rhodothermus obamensis glycogen branching enzyme to enhance those of slowly digestible property. The synthesized α-glucans (Mw: 1.7-4.9 × 107 g mol-1) were mainly composed of α-1,4 linkages and large proportions of α-1,6 linkages (7.5%-9.9%). After treating the enzymatically synthesized α-glucans with the human α-amylase, the quantity of branched α-LDx (36.2%-46.7%) observed was higher than that for amylopectin (26.8%) and oyster glycogen (29.1%). When the synthetic α-glucans were hydrolyzed by mammalin α-glucosidases, the glucose generation rate decreased because the amount of embedded branched α-LDx increased. Therefore, the macro-sized branched α-glucans with high α-LDx has the potential to be used as slowly digestible material to attenuate postprandial glycemic response.

Emerging Trends in Pullulan-Based Antimicrobial Systems for Various Applications.

Global reports on multidrug resistance (MDR) and life-threatening pathogens such as SARS-CoV-2 and Candida cruris have stimulated researchers to explore new antimicrobials that are eco-friendly and economically viable. In this context, biodegradable polymers such as nisin, chitin, and pullulan play an important role in solving the problem. Pullulan is an important edible, biocompatible, water-soluble polymer secreted by Aureobasidium pullulans that occurs ubiquitously. It consists of maltotriose units linked with α-1,6 glycosidic bonds and is classed as Generally Regarded as Safe (GRAS) by the Food and Drug Administration (FDA) in the USA. Pullulan is known for its antibacterial, antifungal, antiviral, and antitumor activities when incorporated with other additives such as antibiotics, drugs, nanoparticles, and so on. Considering the importance of its antimicrobial activities, this polymer can be used as a potential antimicrobial agent against various pathogenic microorganisms including the multidrug-resistant (MDR) pathogens. Moreover, pullulan has ability to synthesize biogenic silver nanoparticles (AgNPs), which are remarkably efficacious against pathogenic microbes. The pullulan-based nanocomposites can be applied for wound healing, food packaging, and also enhancing the shelf-life of fruits and vegetables. In this review, we have discussed biosynthesis of pullulan and its role as antibacterial, antiviral, and antifungal agent. Pullulan-based films impregnated with different antimicrobials such as AgNPs, chitosan, essential oils, and so on, forming nanocomposites have also been discussed as natural alternatives to combat the problems posed by pathogens.

Aspergillus nidulans AmyG Functions as an Intracellular α-Amylase to Promote α-Glucan Synthesis.

α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear. α-Glucan was shown to be composed mainly of 1,3-glycosidically linked glucose, with trace amounts of 1,4-glycosidically linked glucose. Besides the α-glucan synthetases, amylase-like proteins were also important for α-glucan synthesis. In our previous work, we showed that Aspergillus nidulans AmyG was an intracellular protein and was crucial for the proper formation of α-glucan. In the present study, we expressed and purified AmyG in an Escherichia coli system. Enzymatic characterization found that AmyG mainly functioned as an α-amylase that degraded starch into maltose. AmyG also showed weak glucanotransferase activity. Most intriguingly, supplementation with maltose in shaken liquid medium could restore the α-glucan content and the phenotypic defect of a ΔamyG strain. These data suggested that AmyG functions mainly as an intracellular α-amylase to provide maltose during α-glucan synthesis in A. nidulans. IMPORTANCE Short α-1,4-glucan was suggested as the primer structure for α-glucan synthesis. However, the exact structure and its source remain elusive. AmyG was essential to promote α-glucan synthesis and had a major impact on the structure of α-glucan in the cell wall. Data presented here revealed that AmyG belongs to the GH13_5 family and showed strong amylase function, digesting starch into maltose. Supplementation with maltose efficiently rescued the phenotypic defect and α-glucan deficiency in an ΔamyG strain but not in an ΔagsB strain. These results provide the first piece of evidence for the primer structure of α-glucan in fungal cells, although it might be specific to A. nidulans.

The GATA type transcriptional factors regulate pullulan biosynthesis in Aureobasidium melanogenum P16.

Aureobasidium melanogenum P16, the high pullulan producer, had only one GATA type transcriptional activator AreA and one GATA type transcriptional repressor AreB. It was found that 2.4 g/L of (NH4)2SO4 had obvious nitrogen repression on pullulan biosynthesis by A. melanogenum P16. Removal of the AreB gene could make the disruptant DA6 produce 34.8 g/L pullulan while the P16 strain only produced 28.8 g/L pullulan at the efficient nitrogen condition. Further both removal of the native AreA gene and overexpression of the mutated AreAS628-S678 gene with non-phosphorylatable residues could render the transformant DEA12 to produce 39.8 g/L pullulan. The transcriptional levels of most of the genes related to pullulan biosynthesis in the transformant DEA12 were greatly enhanced. The mutated AreAS628-S678 was localized in the nuclei of the transformant DEA12 while the native AreA was distributed in the cytoplasm in A. melanogenum P16. This meant that nitrogen repression on pullulan biosynthesis in the transformant DEA12 was indeed significantly relieved. This was the first time to report that the GATA type transcriptional factors of nitrogen catabolite repression system could regulate pullulan biosynthesis in Aureobasidium spp.

High level expression of a xyloglucanase from Rhizomucor miehei in Pichia pastoris for production of xyloglucan oligosaccharides and its application in yoghurt.

The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL-1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg-1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.

Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis.

Certain Aspergillus and Penicillium spp. produce the fungal cell wall component nigeran, an unbranched d-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear. We used RNA sequencing (RNA-seq) to identify genes involved in nigeran synthesis in the filamentous fungus Aspergillus luchuensis when grown under nitrogen-free conditions. agsB, which encodes a putative α-1,3-glucan synthase, and two adjacent genes (agtC and gnsA) were upregulated under conditions of nitrogen starvation. Disruption of agsB in A. luchuensis (ΔagsB) resulted in the complete loss of nigeran synthesis. Furthermore, the overexpression of agsB in an Aspergillus oryzae strain that cannot produce nigeran resulted in nigeran synthesis. These results indicated that agsB encodes a nigeran synthase. Therefore, we have renamed the A. luchuensis agsB gene the nigeran synthase gene (nisA). Nigeran synthesis in an agtC mutant (ΔagtC) increased to 121%; conversely, those in the ΔgnsA and ΔagtC ΔgnsA strains decreased to 64% and 63%, respectively, compared to that in the wild-type strain. Our results revealed that AgtC and GnsA play an important role in regulating not only the quantity of nigeran but also its polymerization. Collectively, our results demonstrated that nisA (agsB) is essential for nigeran synthesis in A. luchuensis, whereas agtC and gnsA contribute to the regulation of nigeran synthesis and its polymerization. This research provides insights into fungal cell wall biosynthesis, specifically the molecular evolution of fungal α-glucan synthase genes and the potential utilization of nigeran as a novel biopolymer. IMPORTANCE The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3/1,4-glucosidic linkages. The mechanisms regulating the biosynthesis and function of nigeran are unknown. Here, we performed RNA sequencing of Aspergillus luchuensis cultured under nitrogen-free or low-nitrogen conditions. A putative α-1,3-glucan synthase gene, whose transcriptional level was upregulated under nitrogen-free conditions, was demonstrated to encode nigeran synthase. Furthermore, two genes encoding an α-glucanotransferase and a hypothetical protein were shown to be involved in controlling the nigeran content and molecular weight. This study reveals genes involved in the synthesis of nigeran, a potential biopolymer, and provides a deeper understanding of fungal cell wall biosynthesis.

Xyloglucan Xylosyltransferase 1 Displays Promiscuity Toward Donor Substrates During in Vitro Reactions.

Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate xyloglucan xylosyltransferase 1 (XXT1), a GT involved in the synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from uridine diphosphate (UDP)-xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-xylose and UDP-glucose, while reactions with UDP-arabinose and UDP-galactose are over 10-fold slower. Using kcat/KM as a measure of efficiency, UDP-xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting that there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.

Natural products targeting the synthesis of ß(1,3)-D-glucan and chitin of the fungal cell wall. Existing drugs and recent findings.

BACKGROUND: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as ß(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of ß(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. PURPOSE: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. METHODS: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms "natural antifungals" and "plant extracts" with "fungal cell wall". RESULTS: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. CONCLUSION: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa.

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.

Structures, Biosynthesis, and Physiological Functions of (1,3;1,4)-ß-D-Glucans.

(1,3;1,4)-ß-D-Glucans, also named as mixed-linkage glucans, are unbranched non-cellulosic polysaccharides containing both (1,3)- and (1,4)-ß-linkages. The linkage ratio varies depending upon species origin and has a significant impact on the physicochemical properties of the (1,3;1,4)-ß-D-glucans. (1,3;1,4)-ß-D-Glucans were thought to be unique in the grasses family (Poaceae); however, evidence has shown that (1,3;1,4)-ß-D-glucans are also synthesized in other taxa, including horsetail fern Equisetum, algae, lichens, and fungi, and more recently, bacteria. The enzyme involved in (1,3;1,4)-ß-D-glucan biosynthesis has been well studied in grasses and cereal. However, how this enzyme is able to assemble the two different linkages remains a matter of debate. Additionally, the presence of (1,3;1,4)-ß-D-glucan across the species evolutionarily distant from Poaceae but absence in some evolutionarily closely related species suggest that the synthesis is either highly conserved or has arisen twice as a result of convergent evolution. Here, we compare the structure of (1,3;1,4)-ß-D-glucans present across various taxonomic groups and provide up-to-date information on how (1,3;1,4)-ß-D-glucans are synthesized and their functions.

Elucidating carbohydrate metabolism in Euglena gracilis: Reverse genetics-based evaluation of genes coding for enzymes linked to paramylon accumulation.

Euglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytoflagellate can accumulate the carbon photoassimilate as a linear ß-1,3-glucan chain called paramylon. This storage polysaccharide can undergo degradation to provide glucose units to obtain ATP and reducing power both in aerobic and anaerobic growth conditions. Our group has recently characterized an essential enzyme for accumulating the polysaccharide, the UDP-glucose pyrophosphorylase (Biochimie vol 154, 2018, 176-186), which catalyzes the synthesis of UDP-glucose (the substrate for paramylon synthase). Additionally, the identification of nucleotide sequences coding for putative UDP-sugar pyrophosphorylases suggests the occurrence of an alternative source of UDP-glucose. In this study, we demonstrate the active involvement of both pyrophosphorylases in paramylon accumulation. Using techniques of single and combined knockdown of transcripts coding for these proteins, we evidenced a substantial decrease in the polysaccharide synthesis from 39 ± 7 µg/106 cells determined in the control at day 21st of growth. Thus, the paramylon accumulation in Euglena gracilis cells decreased by 60% and 30% after a single knockdown of the expression of genes coding for UDP-glucose pyrophosphorylase and UDP-sugar pyrophosphorylase, respectively. Besides, the combined knockdown of both genes resulted in a ca. 65% reduction in the level of the storage polysaccharide. Our findings indicate the existence of a physiological dependence between paramylon accumulation and the partitioning of sugar nucleotides into other metabolic routes, including the Leloir pathway's functionality in Euglena gracilis.

Correlation between the synthesis of pullulan and melanin in Aureobasidium pullulans.

The content of pullulan and melanin in 500 mutants of Aureobasidum pullulans obtained by ultraviolet mutagenesis were examined and statistically analyzed, and a strong positive correlation was found between them. The result was further confirmed by culturing wild type strain As3.3984 in different media. Then we constructed melanin-deletion mutant As-Δalb1 and pullulan-deletion mutant As-Δpul. As-Δalb1 was a melanin-free strain with the yield of pullulan decreased by 41.01%. The supplementation of melanin in the culture of As-Δalb1 increased the production of pullulan. As-Δpul synthesized neither pullulan nor melanin and recovered melanin synthesis by adding pullulan to the medium. The results suggested that high concentration- of pullulan induced morphological transformation and synthesis of melanin, and melanin promoted the synthesis of pullulan. The pullulan biosynthetic genes, upt, pgm, ugp, and pul, were down-regulated, while the negative regulatory gene of pullulan synthesis, creA, was up-regulated by melanin deficiency.

Polysaccharides Release in a Laboratory-Scale Batch Hydrothermal Pretreatment of Wheat Straw under Rigorous Isothermal Operation.

Hydrothermal pretreatment (HP) is an eco-friendly process for deconstructing lignocellulosic biomass (LCB) that plays a key role in ensuring the profitability of producing biofuels or bioproducts in a biorefinery. At the laboratory scale, HP is usually carried out under non-isothermal regimes with poor temperature control. In contrast, HP is usually carried out under isothermal conditions at the commercial scale. Consequently, significant discrepancies in the values of polysaccharide releases are found in the literature. Therefore, laboratory-scale HP data are not trustworthy if scale-up or retrofitting of HP at larger scales is required. This contribution presents the results of laboratory-scale batch HP for wheat straw in terms of xylan and glucan release that were obtained with rigorous temperature control under isothermal conditions during the reaction stage. The heating and cooling stages were carried out with fast rates (43 and -40 °C/min, respectively), minimizing non-isothermal reaction periods. Therefore, the polysaccharide release results can be associated exclusively with the isothermic reaction stage and can be considered as a reliable source of information for HP at commercial scales. The highest amount of xylan release was 4.8 g/L or 43% obtained at 180 °C and 20 min, while the glucan release exhibited a maximum of 1.2 g/L or 5.5%. at 160 °C/180 °C and 30 min.

Analysis of Structural and Functional Differences of Glucans Produced by the Natively Released Dextransucrase of Liquorilactobacillus hordei TMW 1.1822.

The properties of the glucopolymer dextran are versatile and linked to its molecular size, structure, branching, and secondary structure. However, suited strategies to control and exploit the variable structures of dextrans are scarce. The aim of this study was to delineate structural and functional differences of dextrans, which were produced in buffers at different conditions using the native dextransucrase released by Liquorilactobacillus (L.) hordei TMW 1.1822. Rheological measurements revealed that dextran produced at pH 4.0 (MW = 1.1 * 108 Da) exhibited the properties of a viscoelastic fluid up to concentrations of 10% (w/v). By contrast, dextran produced at pH 5.5 (MW = 1.86 * 108 Da) was gel-forming already at 7.5% (w/v). As both dextrans exhibited comparable molecular structures, the molecular weight primarily influenced their rheological properties. The addition of maltose to the production assays caused the formation of the trisaccharide panose instead of dextran. Moreover, pre-cultures of L. hordei TMW 1.1822 grown without sucrose were substantial for recovery of higher dextran yields, since the cells stored the constitutively expressed dextransucrase intracellularly, until sucrose became available. These findings can be exploited for the controlled recovery of functionally diverse dextrans and oligosaccharides by the use of one dextransucrase type.

Pullulan biosynthesis and its regulation in Aureobasidium spp.

It has been well known that different strains of Aureobasidium spp. can yield a large amount of pullulan. Although pullulan has wide applications in various sectors of biotechnology, its biosynthesis and regulation were not resolved. Lately, the molecular mechanisms of pullulan biosynthesis and regulation have been elucidated and their genes and encoding proteins have been identified using the genome-wide mutant analysis. It is found that a multidomain AmAgs2 is the key enzyme for pullulan biosynthesis and the alternative primers are required for its biosynthesis. Pullulan biosynthesis is regulated by glucose repression and signaling pathways. Elucidation of such a biosynthetic pathway and regulation is of significance in biotechnology. Therefore, the present review article mainly summaries the recent research proceedings in this field, hoping to promote further endeavors on enhanced pullulan production and improved chemical properties of pullulan via molecular modifications of the producers by using synthetic biology approaches.

Glycerol, trehalose and vacuoles had relations to pullulan synthesis and osmotic tolerance by the whole genome duplicated strain Aureobasidium melanogenum TN3-1 isolated from natural honey.

In our previous study, it was found that Aureobasidium melanogenum TN3-1 was a high pullulan producing and osmotic tolerant yeast-like fungal strain. In this study, the HOG1 signaling pathway controlling glycerol synthesis, glycerol, trehalose and vacuoles were found to be closely related to its pullulan biosynthesis and high osmotic tolerance. Therefore, deletion of the key genes for the HOG1 signaling pathway, glycerol and trehalose biosynthesis and vacuole formation made all the mutants reduce pullulan biosynthesis and increase sensitivity of the growth of the mutants to high glucose concentration. Especially, abolishment of both the VSP11 and VSP12 genes which controlled the fission/fusion balance of vacuoles could cause big reduction in pullulan production (less than 7.4 ± 0.4 g/L) by the double mutant ΔDV-5 and increased sensitivity to high concentration glucose, while expression of the VSP11 gene in the double mutant ΔDV-5 made the transformants EV-2 restore pullulan production and tolerance to high concentration glucose. But cell growth of them were the similar. The double mutant ΔDV-5 had much bigger vacuoles and less numbers of vacuoles than the transformant EV-2 and its wild type strain TN3-1 while it grew weakly on the plate with 40% (w/v) glucose while the transformant EV-2 and its wild type strain TN3-1 could grow normally on the plate even with 60% (w/v) glucose. The double mutant ΔDV-5 also had high level of pigment and its cells were swollen. This was the first time to give the evidence that glycerol, trehalose and vacuoles were closely related to pullulan biosynthesis and high osmotic tolerance by A. melanogenum.

Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.

Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.

Cyclodextrinase from Thermococcus sp expressed in Bacillus subtilis and its application in the preparation of maltoheptaose.

BACKGROUND: Maltoheptaose as malto-oligosaccharides with specific degree of polymerization, has wide applications in food, medicine and cosmetics industries. Currently, cyclodextrinase have been applied as prepared enzyme to prepare maltoheptaose. However, the yield and proportion of maltoheptaose was lower, which is due to limited substrate and product specificity of cyclodextrinase (CDase). To achieve higher maltoheptaose yield, cyclodextrinase with high substrate and product specificity should be obtained. RESULTS: In this study, cyclodextrinase derived from Thermococcus sp B1001 (TsCDase) was successfully expressed and characterized in Bacillus subtilis for the first time. The specific activity of TsCDase was 637.95 U/mg under optimal conditions of 90 °C and pH 5.5, which exhibited high substrate specificity for cyclodextrins (CDs). When the concentration of ß-CD was 8%, the yield of maltoheptaose achieved by TsCDase was 82.33% across all reaction products, which exceeded the yields of maltoheptaose in other recent reports. Among malto-oligosaccharides generated as reaction products, maltoheptaose was present in the highest proportion, about 94.55%. CONCLUSIONS: This study provides high substrate and product specificity of TsCDase. TsCDase is able to prepare higher yield of maltoheptaose through conversion of ß-CD in the food industry.