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Bone defect is usually difficult to recover quickly, and bone scaffold transplantation is considered to be an effective method. Biomaterials have a wide range of application prospects in bone tissue repair, and the two key problems are the selection of materials and cells. The object of this study was to discuss the structural characteristics of bone scaffold materials and their effects on bone repair in vivo. The chitin-hydroxyapatite (HAP)-collagen composite scaffolds (CHCS) was prepared with epichlorohydrin (ECH) as crosslinking agent. The structure was characterized and the compressive strength, porosity, water absorbency and stability were investigated. The biocompatibility and osteogenic differentiation of CHCS in vitro were detected, and the effect of defect repair in vivo was evaluated. The results suggested that HAP not only enhanced the compressive strength of CHCS, but also promoted the formation of calcium nodules due to its bone conductivity. Histological staining showed that collagen promoted collagen deposition and new bone formation. X-ray images also indicated that CHCS transplantation accelerated bone repair. Therefore, CHCs has immense potential in bone regeneration.

Many genes responsible for melanin biosynthesis in fungi were physically linked together. The PKS gene clusters in most of the melanin-producing fungi were regulated by the Cmr1. It was found that a close rearrangement of the PKS gene clusters had evolved in most of the melanin-producing fungi and various functions of melanin in them were beneficial to their adaptation to the changing environments. The melanin-producing fungi had undergone at least five large-scale differentiations, making their PKS gene clusters be quickly evolved and the fungi be adapted to different harsh environments. The recent gene losses and expansion were remarkably frequent in the PKS gene clusters, leading to their rapid evolution and adaptation of their hosts to different environments. The PKS gene and the CMR1 gene in them were subject to a strong co-evolution, but the horizontal gene transfer events might have occurred in the genome-duplicated species, Aspergillus and Penicillium.

Several dressing materials can be used efficiently in recent times, both in their natural and synthetic combinations like; microfibers, film, nanofibers, hydrogels, and various drugs. The specific characteristics, such as biocompatibility and providing a favorable environment for wound healing, make many polysaccharides pivotal as wound dressings. Keeping in view the importance of these polysaccharides, we have developed novel chitosan-ulvan hydrogel incorporated by cellulose nanocrystals (CNCs) loading epidermal growth factor (EGF) drug (CS-U-CNC-EGF) by the freeze-dried process. The morphological features of novel hydrogel were perceived by FTIR, XRD, FESEM, and DSC analysis. The incorporation of the nanocrystals content modified the porous microstructure at pore size from 237 ± 59 μm to 53 ± 16 μm, improved mechanical stress curve from 0.57 MPa to 1.2 MPa, thermal and swelling behavior. The novel nanocomposites revealed non-toxic behavior and excellent cell proliferation. Whereas hydrogel showed sustained release of the epidermal growth factor (EGF), thereby enhancing EGF delivery at the wound site for 15 days from a 100% wound contraction treated group. Moreover, the controlled release of EGF from CS-U-CNC-EGF hydrogels showed significantly faster-wound healing efficiency concerning considerably faster granulations tissue formation and collagen deposition. The study's results point to possible future applications of this composite hydrogel in wound healing as a wound dressing material.

It has been well documented that different strains of Aureobasidium spp. can synthesize and secrete over 30.0 g/L of polymalate (PMA) and the produced PMA has many potential applications in biomaterial, medical and food industries. The substrates for PMA biosynthesis include glucose, xylose, fructose, sucrose and glucose or fructose or xylose or sucrose-containing natural materials from industrial and agricultural wastes. Malate, the only monomer for PMA biosynthesis mainly comes from TCA cycle, cytosolic reduction TCA pathway and the glyoxylate cycle. The PMA synthetase (a NRPS) containing A like domain, T domain and C like domain is responsible for polymerization of malate into PMA molecules by formation of ester bonds between malates. PMA biosynthesis is regulated by the transcriptional activator Crz1 from Ca2+ signaling pathway, the GATA-type transcription factor Gat1 from nitrogen catabolite repression and the GATA-type transcription factor NsdD.

In order to increase content of glucuronic acid in the exopolysaccharide (EPS) and its flocculating activity, an UDP-glucose dehydrogenase gene was overexpressed in Lipomyces starkeyi V19. The obtained U9 strain could produce 62.1 ± 1.2 g/l EPS while the V19 strain only produced 53.5 ± 1.3 g/l EPS. The compositions of monosaccharides (mannose, glucuronic acid and galactose) in the purified EPS (U9-EPS) from the U9 strain contained 3.79:1:5.52 while those in the purified EPS (V19-EPS) were 3.94:1:6.29. The flocculation rate of the U9-EPS on kaolin clay reached 87.9%, which was significantly higher than that (74.7%) of the V19-EPS while the decolorization rate of Congo Red (CR) by the U9-EPS reached 94.3%, which was significantly higher than that of CR by the V19-EPS (86.23%). The results showed that the purified bioflocculant U9-EPS had effective flocculation of kaolin clay. The U9-EPS also had high ability to flocculate the polluted river water and decolorize Congo red.

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.

The fungal α-glucan synthases (Agss) are multi-domain proteins catalyzing biosynthesis of cell wall α-1,3-glucan which determines cell wall integrity or fungal pathogenicity and pullulan which is a maltotriosyl polymer made of α-1,4 and α-1,6 bound glucose units. The Agss family can be divided into 11 groups, some of which lost the original functions due to accumulation of harmful mutations or gene loss. Schizosaccharomyces pombe kept five kinds of Agss in the genome while Aspergillus spp. and Penicillium spp. lost one or two or three kinds of Agss. All the human, animal and plant pathogens kept only one single kind of Ags or only one active Ags for synthesis of cell wall α-1,3-glucan, a virulence factor. While the genus Aureobasidium spp. contained three kinds of Agss, of which only some of the Ags2 was involved in pullulan biosynthesis. Although many Agss contained Big_5 domain, only the Big_5 domain with conserved amino acids LQS from some strains of A. melanogenum could catalyze pullulan biosynthesis. This whole amino acid sequence and phylogenetic differences may cause non-α-1,3-glucan synthesizing activity of some fungal Agss.

Pullulan is an important polysaccharide. Although its synthetic pathway in Aureobasidium melanogenum has been elucidated, the mechanism underlying its biosynthesis as regulated by signaling pathway and transcriptional regulator is still unknown. In this study, it was found that the expression of the UGP1 gene encoding UDPG-pyrophosphorylase (Ugp1) and other genes which were involved in pullulan biosynthesis was controlled by the transcriptional activator Msn2 in the nuclei of yeast-like fungal cells. The Ugp1 was a rate-limiting enzyme for pullulan biosynthesis. In addition, the activity and subcellular localization of the Msn2 were regulated only by the cAMP-PKA signaling pathway. When the cAMP-PKA activity was low, the Msn2 was localized in the nuclei, the UGP1 gene was highly expressed, and pullulan was actively synthesized. By contrast, when the cAMP-PKA activity was high, the Msn2 was localized in the cytoplasm and the UGP1 gene expression was disabled so that pullulan was stopped, but lipid biosynthesis was actively enhanced. This study was the first to report that pullulan and lipid biosynthesis in yeast-like fungal cells were regulated by the Msn2 and cAMP-PKA signaling pathway. Elucidating the regulation mechanisms was important to understand their functions and enhance pullulan and lipid biosynthesis.

Mangrove fungi, their ecological role in mangrove ecosystems, their bioproducts, and potential applications are reviewed in this article. Mangrove ecosystems can play an important role in beach protection, accretion promotion, and sheltering coastlines and creeks as barriers against devastating tropical storms and waves, seawater, and air pollution. The ecosystems are characterized by high average and constant temperatures, high salinity, strong winds, and anaerobic muddy soil. The mangrove ecosystems also provide the unique habitats for the colonization of fungi which can produce different kinds of enzymes for industrial uses, recycling of plants and animals in the ecosystems, and the degradation of pollutants. Many mangrove ecosystem-associated fungi also can produce exopolysaccharides, Ca2+-gluconic acid, polymalate, liamocin, polyunsaturated fatty acids, biofuels, xylitol, enzymes, and bioactive substances, which have many potential applications in the bioenergy, food, agricultural, and pharmaceutical industries. Therefore, mangrove ecosystems are rich bioresources for bioindustries and ecology. It is necessary to identify more mangrove fungi and genetically edit them to produce a distinct array of novel chemical entities, enzymes, and bioactive substances.

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.

In this work, a biological engineering-based biosilica-yeast hybrid material was developed. It was obtained by the aggregation of Yarrowia lipolytica through biosilicification catalyzed using genetically displayed silicatein on its cell surface. With orthosilicate or seawater as the substrate, the silicatein-displayed yeast could aggregate into flocs with a flocculation efficiency of nearly 100%. The resulting floc was found to be a sheetlike biosilica-yeast hybrid material formed by the biosilica-mediated immobilization of yeast cells via cross-linking and embedding, turning the original hydrophilicity of yeast cells into hydrophobicity. In addition, this material was characterized to be porous with an average pore diameter of approximately 10 μm and porosity of over 70%. Because of these properties, this hybrid material could achieve enhanced removal efficiencies for chromium ions and n-hexadecane, which were both above 99%, as compared to the free cells of Y. lipolytica in aqueous environments. Importantly, this hybrid material could be recultivated to generate new batches of yeast cells that maintain parallel properties to the first generation so that the same hybrid material could be reproduced with unchanged highly efficient removal of chromium and n-hexadecane to those of the first generation, demonstrating that this biosilica-yeast hybrid material was living and renewable. This work presented a novel way of harnessing silicatein and Y. lipolytica to achieve biological synthesis of a living inorganic-organic hybrid material that has potential to be applied in water treatment.

Although pullulan has many uses in industry, the detailed mechanisms of its biosynthesis still require clarification. In this study, it was found that a short α-1,4-glucosyl chain (pullulan primer) synthesized by the glycogenins Glg1 and Glg2 for initiation of glycogen biosynthesis was also needed for pullulan synthesis. The primers were also synthesized on sterol glycosides and glucosylceramides by catalysis of sterol glucosyltransferase (Sgt1) and ceramide β-glucosyltransferase (Gcs1). All the primers might be elongated to be long α-1,4-glucosyl chain (pullulan precursor) by catalysis of the glycogen synthetase domain of the AmAgs2 as previously reported. Then, the amylase domain of the same AmAgs2 was responsible for pullulan biosynthesis. Removal of all the genes encoding Glg1, Glg2, Gcs1 and Sgt1 made all the mutants produce much less pullulan than the strain P16. However, pullulan synthesis could not be stopped totally in these mutants, suggesting that any other unknown alternative pullulan primers may exist in the yeast cells. Complementation of all the genes in the mutants restored pullulan biosynthesis. This is the first time to report that like starch and glycogen biosynthesis, alternative primers are also required for pullulan biosynthesis in Aureobasidium melanogenum P16.

Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries.

It has been well known that different strains of Aureobasidium spp. can produce commercial pullulan, polymalate, liamocin, intracellular lipids, gluconic acid, siderophore, melanin and various enzymes. In order to fully elucidate their synthetic pathways and regulation, it is necessary to have an efficient gene editing system for genetic modification of Aureobasidium spp. In this study, an efficient Cre/loxp site-specific recombination system (pAMGDloxp-1, pAMEXlox-1 and pAMCRE1) was constructed. It was found that they could be successfully used to sequentially delete and express many genes in different strains of A. melanogenum. After each round of gene disruption and expression, over 0.5 positive cells per 1000 competent cells and over 49.8 positive transformants per 1.0 μg DNA were achieved. After each round of the antibiotics gene excision by using the Cre-loxp site-specific recombination, over 95.4 % of the antibiotics-resistant cells became sensitive to both hygromycin B and nourseothricin again. This demonstrated that the Cre/loxp site-specific recombination system constructed in this study can efficiently be used to simultaneously delete and express many genes in different strains of A. melanogenum. These systems are promising approaches for the easily modifying genomics of the yeast-like fungal strains with enhanced metabolic pathways through multicopy gene deletion and expression.

Background Polyol esters of fatty acids (PEFA) are a kind of promising biosurfactants and mainly secreted by Rhodotorula strains. In addition, some strains of Rhodotorula are reliable producers of microbial lipid. Therefore, it is feasible to establish a one step fermentation process for efficient simultaneous production of PEFA and microbial lipids by a suitable Rhodotorula strain. Results A newly isolated deep-sea yeast, Rhodotorula paludigena P4R5, was shown to simultaneously produce high level of intracellular lipid and extracellular PEFA. Under the optimized conditions, it could yield 48.5 g/L of PEFA and 16.9 g/L of intracellular lipid within 156 h from inulin during 10-L batch fermentation. The PEFA consisting of a mixture of mannitol esters of 3-hydroxy C14, C16 and C18 fatty acids with variable acetylation showed outstanding surface activity and emulsifying activity, while the fatty acids of the intracellular lipid were mainly C16 and C18 and could be high-quality feedstock for biodiesel production. Conclusion The deep-sea yeast strain R. paludigena P4R5 was an excellent candidate for efficient simultaneous of biosurfactants and biodiesel from inulin. Our results also suggested that the establishment of fermentation systems with multiple metabolites production was an effective approach to improve the profitability.

The biomass from Enteromorpha prolifera is used to exploit and transfer this common green bloom algae into new value-added products. In this work, cellulose nanocrystals (CNCs) were first prepared with the cellulose extracted from the biomass of E. prolifera. Initially, cellulose was treated by alkali and bleaching treatments. Subsequently, CNCs were isolated by acid hydrolysis with different concentrations. Structural changes and crystallinity were characterized by Fourier transform infrared spectroscopy (FTIR) spectroscopy, X-ray diffraction (XRD) analysis and t thermogravimetric analysis. The crystallinity index and crystallite size of the prepared CNCs depended mainly on acid concentrations. TEM observation revealed that the CNCs obtained by hydrolysis with 60% acid (CNC60) exhibited a ‘needle-like’ nanocrystals with a length of 177 ± 12 nm and width of 3 ± 1 nm, indicating that the CNC60 had a high ratio aspect and could serve as reinforcement nanofiller. Further investigation showcased that the addition of CNC60 exhibited the best effect for enhancing the mechanical properties of bionanocomposite films derived from the water-soluble polysaccharides extracted from the biomass of E. prolifera. Therefore, a new strategy was offered in this study for the comprehensive utilization of E. prolifera biomass to fabricate them into high-strength film materials.

A novel yeast strain Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert was found to be able to produce a high level of extracellular polysaccharide (EPS). Under the optimal conditions, the yeast strain could yield 73.25 ± 2.3 g/L of EPS within 5 days at a flask level. During a 10-liter fermentation, the yeast strain could produce 78.05 ± 3.5 g/L of EPS within 120 h. The FT-IR spectra of the standard pullulan from Sigma and the purified EPS produced by A. melanogenum 13-2 were almost identical and the purified EPS could be actively hydrolyzed by a pullulanase, demonstrating that the purified EPS was pullulan. The molecular weight (Mw) of the purified pullulan was estimated to be 7.703 × 105 g/moL. Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 lose the ability to synthesize any pullulan. The mutant DAG27 was more sensitive to radiation of UV light, high NaCl concentration, heat treatment, strong oxidation of H2O2 and desiccation than its wild type strain 13-2, suggesting that the produced pullulan could play an important role in resistance of the yeast cells to various stresses. This was the first time to show that the yeast strain obtained from the desert could produce such high level pullulan and the produced pullulan had an obviously protective effect on its producer.

Author: Recently, increasing research in siderophores has been dedicated to their possible medical applications in diagnostics and therapeutics for human pathogenic infections. Fusarinine C (FsC) is a natural hydroxamate siderophore that harbors three amino groups, which allow the easy chemical modification of FsC for the design of novel multifunctional conjugates. However, low production of FsC has hampered its extensive exploitation.Herein, we rewired the FsC biosynthetic pathway in the Aureobasidium melanogenum HN6.2 strain to achieve a self-supplying l-ornithine with component-simplified and enhanced production of extracellular siderophores, for which the FsC accounted for 94%, its final titer being approximately 1.7 g L-1. The convenient acquisition of FsC effectuated our exploitation for its application. We employed in vitro and in vivo assays to show that FsC is an active targeting molecule that acts on the human pathogenic fungi Trichophyton rubrum and Candida albicans; this demonstrates the potential to use FsC for the development of novel antifungal targeting reagents in the future.

This study aimed to overexpress an inulinase gene in Kluyveromyces marxianus to achieve the inulinase overproduction and preparation of ultra-high-fructose syrup. First, the inulinase gene (INU1 gene) was overexpressed through codon optimization and selection of a suitable promoter. Then, the inulinase was overproduced by high-cell-density fermentation. Finally, ultra-high-fructose syrup was prepared. It was found that optimization of the codons of the native INU1 gene encoding inulinase from Kluyveromyces marxianus KM-0 made a recombinant strain KM-N70 carrying the optimized INU1N gene produce 251.4 U/mL of the inulinase activity. Furthermore, inulinase activity produced by a recombinant KM-KN16 strain carrying the optimized INU1N gene directed by the native TPS1 promoter from K. marxianus KM-0 reached 338.5 U/mL and expression level of the optimized INU1N gene in the recombinant KM-KN16 strain was also greatly enhanced. During a 10-L fermentation, the recombinant KM-KN16 strain could produce 374.3 U/mL of inulinase activity within 24 h, while during a high-cell-density fed-batch fermentation, the recombinant KM-KN16 strain could produce 896.1 U/mL of inulinase activity and OD600nm value of its culture reached 108. The crude inulinase preparation obtained in this study had an inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation. It was found that 90.3% of 332.4 g/L of inulin was hydrolyzed to produce 41.0 g/L of glucose and 256.0 g/L of fructose and 91.1% of 328.2 g/L of inulin in the extract of the tubers of Jerusalem artichoke was hydrolyzed to produce 48.3 g/L of glucose and 250.5 g/L of fructose by the crude inulinase preparation (75 U/g of the substrate) within 8 h. The hydrolysates contained major monosaccharides and a trace amount of trisaccharides and the monosaccharides were composed of around 85% fructose and 15% glucose. So far, any other yeasts available have produced only up to 120 U/mL of inulinase activity. Together, this made the recombinant KM-KN16 strain be the best inulinase producer at this moment. The inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation and the ultra-high-fructose syrup with 41.0 g/L of glucose and 256.0 g/L of fructose were obtained. The inulinase activity obtained in this study was the highest among all the inulinase activities produced by yeast, fungal, and bacterial strains obtained so far.

In this study, a new integrative vector (pBCGA) was developed and used for the high-level expression of an optimized ι-carrageenase gene (CGIOP) in Brevibacillus choshinensis. The pBCGA vector allowed multiple copies of the gene CGIOP to be stably integrated into the genomic DNA of B. choshinensis. The recombinant strain I24 could produce an extracellular ι-carrageenase activity of 38.9 U/mL within 72 h, which remained stable after five sequential inoculations and cultivations under the antibiotic-free culture conditions. Furthermore, the strain I24 was applied to 10-L fermentation under the antibiotic-free culture condition, resulting in the highest observed ι-carrageenase activity of 182.4 U/mL within 24 h. Subsequently, recombinant ι-carrageenase (rCgiA) was purified and characterized, exhibiting an optimal pH and temperature of 8.0 and 45 °C, respectively. Notably, rCgiA showed considerable stability below 45 °C and over a relatively broad pH range of 6.0–11.0. In addtion, the activity of rCgiA was significantly stimulated by NaCl, Mg2+, and Ca2+. HILIC LC-LTQ-Orbitrap-FTMS analysis revealed that rCgiA hydrolyzed ι-carrageenan via a processive mechanism with the major product of ι-carrageenan tetrasaccharide. Thus, the strain B. choshinensis I24 had broad potential for use in the environment-friendly and large-scale production of ι-carrageenase and ι-carrageenan oligosaccharides.

Summary Currently, piperazic acid is chemically synthesized using ecologically unfriendly processes. Microbial synthesis from glucose is an attractive alternative to chemical synthesis. In this study, we report the production of L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum; this strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring and constitutive genetic circuit reconstitution, to build a robust L‐piperazic acid synthetic cascade. These genetic modifications enable A. melanogenum to directly convert glucose to L‐piperazic acid without relying on the use of either chemically synthesized precursors or harsh conditions. This bio‐based process overcomes the shortcomings of the conventional synthesis routes. The ultimately engineered strain is a very high‐efficient cell factory that can excrete 1.12 ± 0.05 g l‐1 of L‐piperazic acid after a 120‐h 10.0‐l fed‐batch fermentation; this is the highest titre of L‐piperazic acid reported using a microbial cell factory., L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum was achieved. This strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring, and constitutive genetic circuit reconstitution to build a robust L‐piperazic acid synthetic cascade. The ultimately engineered strain has the highest titer of L‐piperazic acid of 1.12 ± 0.05 g l‐1 ever reported.

TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase., Author summary Toll/interleukin-1 receptor domain-containing protein encoded by E. coli (TcpC) is an important virulence factor in many strains of uropathogenic E. coli (UPEC). TcpC-mediated evasion of innate immunity plays an important role in the pathogenesis of UPEC caused urinary tract infection (UTI) including pyelonephritis. In the present study, we show TcpC is an E3 ubiquitin ligase that promotes ubiquitination and degradation of MyD88, hereby blocking the TLR signaling pathway. Our findings not only illuminate the novel biochemical mechanisms underlying TcpC-mediated evasion of innate immunity, but also provide the first example that bacterial pathogens can subvert TLR signaling pathway through virulence factors that function as MyD88-targeted E3 ubiquitin ligase.

In the present experimental series, we have developed a novel nanocomposite to target activated macrophages in the colon with real time imaging and therapeutic capabilities. This binary nanocomposite was formed by the covalent conjugation of mannosylated NPs (Man-NPs) with carbon dots (CDs). Man-NPs were prepared using a self-assembly method based on mannosylated decamethylenediamine-grafted carboxymethyl inulin amphiphilic acid. While, the CDs were synthesized using a simple bottom-up process using citric acid monohydrate and diethylenetriamine, which were tightly bonded to the Man-NPs surface by carbodimide coupling. The resulting nanocomposite had a uniform size of 241.3 nm with a negative charge and a high drug casing density of 25.54 wt% and blue self-fluorescence were emitted. Whereas, in vitro observation of cellular uptake indicated the greater nanocomposite uptake in inflamed macrophage as compared to the untreated macrophage and mannose receptor-negative cell lines, 4T1 respectively. However, in vivo bio distribution exhibited a large number (60%) of CDs/Man-NPs nanocomposite accumulated in the inflamed colon of colitis mice. It should be noted that the novel nanocomposite, as macrophage-targeted drug delivery, could have promise for the treatment of inflammatory bowel disease (IBD).

The pathogenic yeast strain LIAO causing the milky disease in the Chinese mitten crab belonged to one member of Metschnikowia bicuspidate which could grow well at different temperatures from 28 to 4 °C. It was also found that the pathogenic yeast strain LIAO could grow in the extracts of the muscle, gill, heart tissues, intestinal tracts of the healthy Chinese mitten crabs by using the reducing sugars, amino acids and other nutrients in them. Massoia lactone released from liamocins produced by Aureobasidium melanogenum had high anti-fungal activity against the pathogenic yeast strain LIAO and M. bicuspidate WCY isolated from the diseased marine crabs. The minimal inhibitory concentrations (MIC) and the minimal fungicidal concentration (MFC) in the liquid culture against the pathogenic yeast strain LIAO were 0.15 mg/mL and 0.34 mg/mL, respectively. Massoia lactone as a bio-surfactant could damage the cell membrane, even break the whole cells of the pathogenic yeast strain LIAO and cause cellular necrosis of the pathogenic yeast LIAO. Therefore, Massoia lactone could be used to effectively kill the pathogenic yeast strains and as an effectitve treatment for milky disease in the Chinese mitten crab.

TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion., TcpC is a well characterised multifunctional virulence factor expressed by uropathogenic Eschericia coli. Here the authors show that TcpC also targets neutrophil NETosis via its E3 ligase functionality promoting the degradation of PAD4, and represents an additional immune evasion function of this bacterially derived virulence factor.

In this study, it was found that a Cre/loxP system could be successfully used as a tool for editing the genome of the psychrophilic yeast Metschnikowia australis W7-5 isolated from Antarctica. The deletion and over-expression of the TPS1 gene for trehalose biosynthesis, the GSY gene for glycogen biosynthesis, and the GPD1 and GPP genes for glycerol biosynthesis had no influence on cell growth of the mutants and transformants compared to cell growth of their wild-type strain M. australis W7-5, indicating that trehalose, glycogen, and glycerol had no function in growth of the psychrophilic yeast at different temperatures. However, removal of the SLT2 gene encoding the mitogen-activated protein kinase in the cell wall integrity (CWI) signaling pathway and the SWI4 and SWI6 genes encoding the transcriptional activators Swi4/6 had the crucial influence on cell growth of the psychrophilic yeast at the low temperature, especially at 25 °C and expression of the genes related to cell wall and lipid biosynthesis. Therefore, the cell wall could play an important role in growth of the psychrophilic yeast at different temperatures and biosynthesis of cell wall was actively regulated by the CWI signaling pathway. This was the first time to show that the genome of the psychrophilic yeast was successfully edited and the molecular evidences were obtained to elucidate mechanisms of low temperature growth of the psychrophilic yeast from Antarctica.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97 Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GA g/l and 2480 U/mg of Gox activity. The productivity of Ca2+-GA was 2.78 g/l/h and the yield was 1.1 g/g.

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca2+-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca2+-GA (160.5 ± 5.6 g/L) and higher GOD1 activity (1438.6 ± 73.2 U/mg of protein) than its parent P6 strain (118.7 ± 4.3 g/L of Ca2+-GA and 1100.0 ± 23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8 ± 6.0 g/L of Ca2+-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca2+-GA (186.8 ± 6.0 g/L) than its wild-type strain P6.

Background Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect pullulan and glycogen biosynthesis. Methods The genes related to cell wall integrity were cloned, characterized, deleted and complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and gene expression were examined. Results In this study, the GT6 and GT7 genes encoding different α1,2 mannosyltransferases in Aureobasidium melanogenum P16 were cloned and characterized. The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family. The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphate glucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. General significance This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis in the pullulan producing yeast.

Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article.Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids.It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.

In this study, the gene of the catalytic domain of the κ-carrageenase from Pseudoalteromonas porphyrae LL1 ( PpCGKCD ) was efficiently expressed in Brevibacillus choshinensis . Under the optimized conditions, the highest activity of the extracellular κ-carrageenase reached to 51.5 ± 1.2 U/mL with 96 h. Subsequently, the catalytic domain of the κ-carrageenase (PpCgkCD) was purified and detected by SDS−PAGE, indicating that the PpCgkCD had a molecular weight of 32 kDa. The optimal pH and temperature of the PpCgkCD were 8.0 and 40 °C, respectively. Notably, the PpCgkCD showed considerable stability below 35 °C and over a relatively broad range of alkaline pHs, even at pH of 11.0 for 12 h. Besides, 100.0 mM of Mg 2+ could dramatically enhance the activity of the PpCgkCD by 97.5%. TLC and ESI-Q-TOF MS/MS analysis revealed that the enzymatic hydrolysates were composed of An-G4S-type neocarrabiose units, and the end-products were neo-κ-carrabiose and neo-κ-carratetraose. Furthermore, the PpCgkCD could be employed to efficiently hydrolyze κ-carrageenan via a random non-processive mechanism for the production of a series of even-numbered κ-carrageenan oligosaccharides. This study demonstrated that the PpCgkCD secreted by B. choshinensis had great potential in the commercial production of bioactive oligosaccharide from κ-carrageenan.

An aptamer-based fluorescence assay for culture-independent detection of Pseudomonas aeruginosa was developed. This assay was enabled by highly specific aptamers conjugated with photoluminescent carbon dots (CDs) as the fluorescent probe and graphene oxide (GO) as the quencher. Specially, high-throughput sequencing was achieved during systematic evolution of ligands via exponential enrichment (SELEX) for accurate recognition of aptamers. This assay displayed high specificity towards P. aeruginosa and was resistant to interference by other ubiquitous bacteria including Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Clostridium perfringens. After the conditions were optimized, this assay achieved a wide detection range for P. aeruginosa varying from 101 CFU mL−1 to 107 CFU mL−1. Notably, it approached an excellent detection limit as low as 9 CFU mL−1. Therefore, this fluorescence assay was considered successfully developed for highly sensitive detection of P. aeruginosa. This assay also detected the contamination of P. aeruginosa in tap water and commercial bottled water, thereby suggesting its potential application in real water samples.

A purified polysaccharide from Enteromorpha prolifera (PEP) with a molecular mass of 147.8kDa, was prepared by hot-water extraction method and purified by anion-exchange chromatography and gel filtration chromatography. Low Mw polysaccharide of E. prolifera (LEP, 44.8kDa) was obtained by enzymatic degradation. Sulfated derivatives of the two different Mw polysaccharides were synthesized by chlorosulfonic acid/pyridine method, and the degrees of substitution of SPEP and SLEP were 0.57 and 0.81, respectively. Physicochemical properties and FT-IR spectra showed that enzymatic degradation and sulfated modification of polysaccharides were successful. 13C NMR analysis showed that the sulfate groups mainly attached to C-6 of rhamnose. Moreover, the study revealed that enzymatic degradation and sulfated modification of polysaccharides improved significantly superoxide, hydroxyl and DPPH radicals scavenging activities in vitro, and enhanced obviously moisture absorption/retention capacities. Therefore, these results demonstrated that molecular weight and sulfate groups had obvious effects on antioxidant activities and moisture absorption/retention abilities of the polysaccharides from E. prolifera.

Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

Melanin plays an important role in the stress adaptation of Aureobasidium melanogenum XJ5-1 isolated from the Taklimakan desert. A trehalose-6-phosphate synthase gene (TPS1 gene) was cloned from K5, characterized, and then deleted to determine the role of trehalose in the stress adaptation of the albino mutant K5. No stress response element and heat shock element were found in the promoter of the TPS1 gene. Deletion of the TPS1 gene in the albino mutant rendered a strain DT43 unable to synthesize any trehalose, but DT43 still could grow in glucose, suggesting that its hexokinase was insensitive to inhibition by trehalose-6-phosphate. Overexpression of the TPS1 gene enhanced trehalose biosynthesis in strain ET6. DT43 could not grow at 33 °C, whereas K5, ET6, and XJ5-1 could grow well at this temperature. Compared with K5 and ET6, DT43 was highly sensitive to heat shock treatment, high oxidation, and high desiccation, but all the three strains demonstrated the same sensitivity to UV light and high NaCl concentration. Therefore, trehalose played an important role in the adaptation of K5 to heat shock treatment, high oxidation, and high desiccation.

A convenient and efficient two-stage bioprocess was established for fructo-oligosaccharides (FOSs) production, during which the endo-inulinase was first produced and subsequently the inulin supplemented was directly hydrolyzed by the produced endo-inulinase, in the meantime the generated non-prebiotic saccharides was assimilated by the yeast cells. This process was implemented by an engineered Yarrowia lipolytica strain Enop56, in which an optimized endo-inulinase gene from Aspergillus niger was overexpressed. When the strain Enop56 was fermented with an inulin concentration of 600g/L in a 10-L bioreactor, the inulinase activity, FOSs titer, yield and productivity reached to 551.6U/mL, 546.6g/L, 0.91gFOS/gInulin, and 15.18g/L/h, respectively. Besides, the hydrolysis products were mainly FOSs with polymerization degrees of 3-5 and the total amount of non-prebiotic mono- and disaccharides was only 4.97% in the final fermentation broth. This study demonstrated that the two-stage bioprocess using the strain Enop56 was a promising strategy to produce FOSs on an industrial scale.

After both a lactase gene (LAC4) and its transcriptional activator gene (LAC9) were expressed in Kluyveromyces marxianus KM-69 which was a glucose-derepressed mutant, one transformant called KM-L9-20 grown in an optimized whey medium for lactase production could produce 77.8 ± 4.0 U/ml of lactase activity at a flask level within 36 h. At the same time, expression of the LAC4 gene and its transcriptional activator gene in the transformant KM-L9-20 were also greatly enhanced compared to that of the LAC4 gene and the LAC9 gene in its parent strain K. marxianus KM-69. 157.9 ± 3.2 U/ml of lactase activity was yielded by the transformant KM-L9-20 within 84 h during a batch fermentation while 274.7 ± 4.3 U/ml of lactase activity was produced within 48 h during a fed-batch fermentation. The produced lactase could actively transform lactose into the galacto-oligosaccharides which contained mainly trisaccharides. The optimal conditions for the transgalactosylation reaction were that the lactose concentration, the reaction temperature, pH and the lactase dose were 400 g/l of lactose, 40 °C, 6.5 and 25 U/g of lactose, respectively. Therefore, the transformant KM-L9-20 and the produced lactase had highly potential applications in biotechnology.

After the compositional change of a pullulan production medium, a molecular weight (Mw) of the pullulan produced by Aureobasidium melanogenum P16 was 2.32 × 10 6 and a pullulan titer was 44.4 g/L while a Mw of the pullulan produced by A. melanogenum P16 grown in the initial medium was only 3.47 × 10 5 and a pullulan titer was 65.3 g/L. The increased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase while the decreased pullulan titer was related to the decreased transcriptional levels of the genes encoding 6-P-glucose kinase, glucosyltransferase, α-phosphoglucose mutase, UDPG-pyrophosphorylase and pullulan synthetase. During the 10-L fermentation, when the yeast strain P16 was grown in the initial medium, the pullulan and oligosaccharide titers were 65.5 g/L and 7.8 g/L, respectively and the Mw of the produced pullulan was 4.42 × 10 5 while when the yeast strain P16 was grown in the compositionally changed medium, the pullulan and oligosaccharide titers were 46.4 g/L and 27.8 g/L, respectively and the Mw of the produced pullulan was 2.6 × 10 6 . Most of the oligosaccharides produced by the yeast strain P16 cultivated in the compositionally changed medium had degree of polymerization of 4 and 5. Therefore, both of the high Mw pullulan and oligosaccharides with high levels were produced by the yeast strain P16.

A PKS1 gene responsible for the melanin biosynthesis and a NPG1 gene in Aureobasidium melanogenum XJ5-1 were cloned and characterized. An ORF of the PKS1 gene encoding a protein with 2165 amino acids contained 6495bp while an ORF of the NPG1 gene encoding a protein with 340 amino acids had 1076bp. After analysis of their promoters, it was found that expression of both the PKS1 gene and the NPG1 gene was repressed by nitrogen sources and glucose, respectively. The PKS deduced from the cloned gene consisted of one ketosynthase, one acyl transferase, two acyl carrier proteins, one thioesterase and one cyclase while the PPTase belonged to the family Sfp-type. After disruption of the PKS1 gene and the NPG1 gene, expression of the PKS1 gene and the NPG1 gene and the melanin biosynthesis in the disruptants K5 and DP107 disappeared and expression of the PKS1 gene in the disruptant DP107 was also negatively influenced. However, after the NPG1 gene was complemented in the disruptant DP107, the melanin biosynthesis in the complementary strain BP17 was restored and expression of the PKS1 gene and the NPG1 gene was greatly enhanced, suggesting that the PKS was indeed activated and regulated by the PPTase and expression of the PKS1 gene and the NPG1 gene had a coordinate regulation.

Aureobasidium melanogenum HN6.2 was found to able to produce an esterase of 208.1 ± 2.7 units per ml within 72 h. A molecular weight of the purified esterase was 60.2 kDa and its PI was 4.86. The optimal pH and temperature of the purified esterase were 8.0 and 40 °C, respectively. The purified esterase was stable at the temperature less than 40 °C and in the pH range of 7.5–8.0. The esterase activity was greatly inhibited in the presence of Zn 2+ , Hg 2+ , Fe 2+ , Ni 2+ and SDS. K m and V max values of the enzyme for ρ-nitrophenyl butyrate were 68.6 μM and 251.4 μM per min, respectively. Mass analysis showed that it belonged to a carboxylesterase. After an esterase gene ( Car-Est ) cloned from the genomic DNA of the marine yeast strain HN6.2 was disrupted, the disruptant Y44 obtained exhibited a significant decrease in esterase activity. Meanwhile, a transcriptional level of the Car-Est gene of the disruptant Y44 was only 5.18% that of the Car-Est gene of its wild type strain HN6.2, confirming that the cloned esterase gene was indeed closely related to the esterase activity of the yeast HN6.2 strain.

It has been well known that poly(β-l-malic acid)(PMA) has many potential applications. However, it is still completely unknown how PMA is biosynthesized in Aureobasidium spp. In this study, it was found that malic acid from TCA cycle was the main source for PMA biosynthesis. Especially, the novel PMA synthetase encoded by the PMAs gene, a non-ribosomal peptide synthetase (NRPS) containing A like domain, T domain and C like domain was the key enzyme for polymerization of malate into PMA. Therefore, abolishment of the PMAs gene encoding the novel PMA synthetase rendered the mutant ΔPMAs-3 totally to lose the ability to synthesize any PMA and complementation of the PMAs gene partially restored PMA biosynthesis, but the mutant could grow normally on the YPD plate and in the PMA medium with CaCO3. The transcriptional activator Crz1 in the Ca2+-signal pathway controlled expression of the PMAs gene and PMA biosynthesis. The complete elucidation of the PMA biosynthesis pathway and its regulation was of significant for a deeper understanding of detailed yeast-like fungal PMA synthesis, metabolic engineering and molecular editing for modifying PMA biosynthesis and its physicochemical properties.

Pullulan, a biological macromolecule, has many applications. However, it is completely unknown how and where it is synthesized. In this study, it was found that the multidomain AmAgs2 (α-glucan synthase 2) encoded by an AmAGS2 gene in Aureobasidium melanogenum P16 contained the amylase domain (Amy_D), the glycogen synthetase domain (Gys_D) and the transmembrane regions in which the exopolysaccharide transporter domain (EPST_D) was embedded. Removal of the AmAGS2 gene in A. melanogenum P16 rendered the disruptants not to synthesize any pullulan and complementation of the AmAGS2 gene in the disruptants restored pullulan synthesis. Overexpression of the gene in Aureobasidium melanogenum CBS105.22, a non-pullulan producer, resulted in the transformants producing pullulan. Therefore, the AmAGS2 gene was the key gene responsible for pullulan biosynthesis in A. melanogenum P16. It was speculated that the short α-1,4-glucosyl chains (pullulan primers) were elongated by the Gys_D of the AmAgs2 to form long α-1,4-glucosyl chains (precursors of pullulan). All the precursors were transported to outside plasma membrane by the EPST_D in the transmembrane regions of the AmAgs2. Then, the Amy_D of the AmAgs2 was responsible for both hydrolysis of the endo-α-1,4-linkages in the precursors to release maltotriose and transfer of the maltotriose to Lph-glucose to form α-1,6 glucosidic bonds between maltotrioses in pullulan molecule. This is the first time to report that the AmAgs2 can play the key role in pullulan biosynthesis.

The melanin produced by Aureobasidium melanogenum XJ5-1 obtained from the Taklimakan Desert can play an important role in adaptation of the yeast strain to various stress treatments. It is very important to know how the desert-derived yeast sense, respond and adapt to the harsh environments. However, it is still unclear how melanin is genetically controlled by signaling pathways and transcriptional factors. In this study, it was found that the mitogen-activated protein kinase (MAPK) Slt2 in the cell wall integrity (CWI) signal pathway could regulate activity of the transcriptional activator Swi4; in turn, the Swi4 could control the expression of the CMR1 gene. The melanin-specific transcriptional activator Cmr1 encoded by the CMR1 gene was specifically bound to the promoter with the sequence TTCTCTCCA of the PKS1 gene and strongly stimulated expression of the PKS1 gene and any other genes responsible for melanin biosynthesis, so that a large amount of melanin could be produced by A. melanogenum XJ5-1. Therefore, melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 was controlled mainly by the CWI signal pathway among the cell wall-related signal pathways via a transcriptional activator Cmr and regulation of the melanin biosynthesis in A. melanogenum XJ5-1 was completely different from that of the melanin biosynthesis in any other fungi. This is the first time to show that melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1. This would provide the fundamentals for further research on the desert-derived yeast to sense, respond and adapt to the harsh environments.

Overexpression of the optimized Vitreoscilla hemoglobin (VHb) gene and the native flavohemoglobin (FHb) gene in Aureobasidium melanogenum P16 rendered a V6 strain and a F44 strain to overproduce pullulan compared to that produced by their wild type strain P16. The capacity to bind CO and oxygen in the V6 strain and the F44 strain was also obviously enhanced. At the same time, the transcriptional levels of the relevant genes were also increased in the V6 strain and the F44 strain and the fused vgbop + the gene encoding GFP and FHb gene + the gene encoding GFP were also actively expressed. During a 10-liter fermentation, the P16 strain produced only 72.0 ± 1.0 g/L pullulan, the yield was 0.77 g/g of sucrose, the productivity was 0.5 ± 0.01 g/L/h and only 79.4% of the total sugar was used. In contrast, the strain V6 yielded 102.3 ± 1.8 g/L of pullulan, the yield was 0.89 g/g of sucrose, the productivity was 0.7 ± 0.01 g/L/h and 96.0% of total sugar was used while 101.4 ± 2.9 g/L of pullulan was accumulated in the culture of the strain F44, the yield was 0.88 g/g of sucrose, the productivity was 0.7 ± 0.02 g/L/h and 96.4% of total sugar was utilized. These data strongly demonstrated that the concentration of pullulan, yield, productivity and sugar utilization were greatly enhanced by overexpression of the VHb and FHb. But their cell growth was almost the similar.

Aureobasidium melanogenum P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the CREA gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding α-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93 ± 1.33 g/l pullulan from 120 g/l of glucose, while its wild-type strain P16 produced only 52.0 ± 1.95 g/l pullulan within 132 h. After the CREA gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in A. melanogenum P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering.

After screening of over 300 yeast strains isolated from the mangrove ecosystems, it was found that Aureobasidium sp. P6 strain had the highest inulin-hydrolyzing activity. Under the optimal conditions, this yeast strain produced an inulin-hydrolyzing activity of 30.98 ± 0.8 U/ml after 108 h of a 10-l fermentation. After the purification, a molecular weight of the enzyme which had the inulin-hydrolyzing activity was estimated to be 47.6 kDa, and the purified enzyme could actively hydrolyze both sucrose and inulin and exhibit a transfructosylating activity at 30.0 % sucrose, converting sucrose into fructooligosaccharides (FOS), indicating that the purified enzyme was a β-D-fructofuranosidase. After the full length of a β-D-fructofuranosidase gene (accession number KU308553) was cloned from Aureobasidium sp. P6 strain, a protein deduced from the cloned gene contained the conserved sequences MNDPNGL, RDP, ECP, FS, and Q of a glycosidehydrolase GH32 family, respectively, but did not contain a conserved sequence SVEVF, and the amino acid sequence of the protein from Aureobasidium sp. P6 strain had a high similarity to that of the β-fructofuranosidase from any other fungal strains. After deletion of the β-D-fructofuranosidase gene, the disruptant still had low inulin hydrolyzing and invertase activities and a trace amount of the transfructosylating activity, indicating that the gene encoding an inulinase may exist in the Aureobasidium sp. P6 strain.

The yeast strain XJ5-1 isolated from the Taklimakan desert soil was identified to be a strain of Aureobasdium melanogenum and could produce a large amount of melanin when it was grown in the PDA medium, but its melanin biosynthesis and expression of the PKS gene responsible for the melanin biosynthesis was significantly repressed in the presence of (NH4)2SO4. However, A. melanogenum P5 strain isolated from a mangrove ecosystem grown in both the presence and the absence of (NH4)2SO4 did not produce any melanin. The cell size of A. melanogenum XJ5-1 strain was much higher than that of A. melanogenum P5 strain. The melanized cells of the yeast strain XJ5-1 had higher tolerance to UV radiation, oxidation (200.0 mM H2O2), heat treatment (40 °C), salt shock (200.0 g/L NaCl), desiccation and strong acid hydrolysis (6.0 M HCl) at high temperature (80 °C) than the non-melanized cells of the same yeast strain XJ5-1. At the same time, the melanized cells of the yeast strain XJ5-1 also had higher tolerance to UV radiation, oxidation (200.0 mM H2O2), desiccation and strong acid hydrolysis (6.0 M HCl) at high temperature (80 °C) than A. melanogenum P5 strain, but had similar resistance to heat treatment (40 °C) and salt shock (200.0 g/L NaCl) compared to those of A. melanogenum P5 strain. All the results revealed that many characteristics of A. melanogenum XJ5-1 isolated from the Taklimakan desert soil was different from those of A. melanogenum P5 strain isolated from the mangrove ecosystem.

An exo-inulinase gene from Kluyveromyces marxianus and an endo-inulinase gene from Aspergillus niger were cloned and expressed in Yarrowia lipolytica, respectively. Then, the purified recombinant exo-inulinase (rEXINU) and endo-inulinase (rENINU), either individual or combination with another, were used for hydrolysis of inulin and inulin in tuber meal of Jerusalem artichoke. After the analysis of the dynamic hydrolysis process, it was found that the rEXINU and the rENINU acted synergistically with each other, and the maximum degree of synergistic effect on the level of fructose formation (DSE Fru ) could be obtained with a molar ratio of the rEXINU to the rENINU at 1:1. Furthermore, the investigation of the hydrolysis product in the synergistic hydrolysis process showed that a wide range of oligosaccharides were first generated and then converted to fructose. Based on the enzyme kinetics, substrate specificity and product inhibition of the rEXINU and the rENINU, a synergism mechanism of the them for the inulin hydrolysis was proposed. During the process of the synergistic action, the rENINU preferentially degraded the inulin with high DP, but was inhibited by the produced oligosaccharides. Then the released oligosaccharides which were the optimum substrates of the rEXIUN were rapidly hydrolyzed by the enzyme to produce fructose and glucose. Meanwhile, this process relieved the inhibition of the produced oligosaccharides to the rENINU. Besides, in the synergistic hydrolysis of the inulin in tuber meal of Jerusalem artichoke, the depolymerization of the rENINU could result in the increase of the subtrate solubility to improve the hydrolysis efficiency. Therefore, this kind of the synergistic effect between the exo-inulinase and the endo-inulinase may provide a potential driving force in the industrial application of inulin and inulin-containing materials.

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Poly(β-L-malic acid) is one natural biopolymer that has the outstanding features of biocompatibility, biodegradability, water solubility, and non-immunogenicity, and it is easily chemically modified. So poly(β-L-malic acid) (PMLA) and its derivatives may have a great potential application as a novel drug delivery system and in the production of advanced biomaterials which have attracted so much research attention. The fungi of Aureobasidium spp. have been discovered to be the most suitable candidates for PMLA production in large quantities which satisfy the demand of either research or industry. In this review, we will give an overall summary about the PMLA produced by Aureobasidium spp. based on related research in the last decades and the elaboration of this PMLA producer will also be accomplished. More importantly, the latest proceedings will be specified and some suggestions to the elucidation of a PMLA biosynthesis pathway which remains undefined up to date will be proposed. Finally, through this review, the further exploitation for the application of PMLA from Aureobasidium spp. can be emphasized and promoted.