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In order to know the function of C

Polymalate (PMA) produced by the whole genome duplicated strain Aureobasidium melanogenum OUC had a high molecular weight (Mw) of 3.9 × 10

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.

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.

Liamocins synthesized by Aureobasidium spp. are glycolipids composed of a single mannitol or arabitol headgroup linked to either three, four or even six 3,5-dihydroxydecanoic ester tail-groups. The...

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.

It has been confirmed that Fad12 (∆12 fatty acid desaturase) and Fad15 (∆15 fatty acid desaturase) were responsible for the synthesis of linoleic acid (C18:2, ∆9,12) and linolenic acid (C18:3, ∆9,12,15), respectively. In order to know their function in cold growth of the psychrophilic yeast Metschnikowia australis W7-5, the FAD12 gene and FAD15 gene were deleted, respectively. The intracellular linoleic acid percentage of the obtained Δfad12 mutant was decreased from 27.1–1.5% while the percentage of C18:1 fatty acid was increased from 28.3–55.7%. The growth rate of the Δfad12 mutant was significantly reduced when it was cultured at 5 ℃ and 25 ℃ compared with that of the wild type strain W7-5 under the same conditions. But at 15°C, the mutant grew as well as its wild type strain W7-5. Although C18:3 fatty acid of the Δfad15 strain were not detected, there was no significant difference between the growth of Δfad15 and that of the W7-5 strain at different temperatures. After the FAD12 gene was supplemented, the growth at different temperatures and intracellular fatty acid compositions of the supplementing strain were restored compared to those of the strain W7-5. These results suggested that only linoleic acid synthesized by the psychrophilic yeast, not linolenic acid synthesized, played an important role in adaptation to low temperature (5°C) and high temperature (25°C) of the psychrophilic yeast, Meanwhile, it was found that cell wall in the mutant Δfad12 grown at 5 and 25°C was also negatively affected after the mutant could not synthesize C18:2 fatty acids. This caused the reduced cell growth rate of the mutant Δfad12 grown at 5 and 25°C.

The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming. In this study, the engineered strains of

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.

Lipomyces starkeyi has been regarded as one of the model oleaginous yeasts. However, in this study, Lipomyces kononenkoae BF1S57 strain was found to be able to yield 60.06% (w/v) lipids (11.3 g/L lipids) from 90.0 g/L glucose, its cell mass was 18.8 ± 0.3 g/L within 96 h of a 10-L fermentation. Furthermore, the genome of L. kononenkoae has two copies of the genes encoding the β-subunit and α-subunit of fatty acid synthase, respectively. This may be related to high lipid contents in its cells. Over 94% of the fatty acids of the extracted lipids produced by L. kononenkoae BF1S57 strain was C16:0, C16:1, C18:0, C18:1 and C18:2 fatty acids. The properties of the prepared biodiesels in this study totally met the requirements of US biodiesel ASTM D6751 and EU biodiesel EN 14214. All these results demonstrated that the produced cellular lipids could be used as raw materials for biofuel production.

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.

Liamocins and Massoia lactone have many applications. In this study, the glucose-derepressed mutant Δcrea5 in which the CREA gene was removed could produce 36.5 g/L of liamocins. Furthermore, overexpression of the MSN2 gene in the mutant Δcrea5 made the transformant M60 produce 41.4 g/L of liamocins and further overexpression of the GAL1 gene in the transformant M60 rendered the transformant G40 to produce 49.5 ± 0.4 g/L of liamocins during the 10-L fermentation while their wild type strain 9-1 made only 26.3 g/L of liamocins. The expressed transcription activators Msn2 and Gal1 were localized in the nuclei, promoting expression of the genes responsible for liamocins biosynthesis and sugar transport. Massoia lactone prepared from the produced liamocins could actively kill the spores of the pathogenic fungi from the diseased human skin by inhibiting spore germination and causing cellular necrosis of the fungal spores.

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9-1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.

Purpose This study aimed to look insights into taxonomy, evolution, and biotechnological potentials of a yeast-like fungal strain P6 isolated from a mangrove ecosystem. Methods The genome sequencing for the yeast-like fungal strain P6 was conducted on a Hiseq sequencing platform, and the genomic characteristics and annotations were analyzed. The central metabolism and gluconate biosynthesis pathway were studied through the genome sequence data by using the GO, KOG, and KEGG databases. The secondary metabolite potentials were also evaluated. Results The whole genome size of the P6 strain was 25.41Mb and the G + C content of its genome was 50.69%. Totally, 6098 protein-coding genes and 264 non-coding RNA genes were predicted. The annotation results showed that the yeast-like fungal strain P6 had complete metabolic pathways of TCA cycle, EMP pathway, pentose phosphate pathway, glyoxylic acid cycle, and other central metabolic pathways. Furthermore, the inulinase activity associated with β-fructofuranosidase and high glucose oxidase activity in this strain have been demonstrated. It was found that this yeast-like fungal strain was located at root of most species of Aureobasidium spp. and at a separate cluster of all the phylogenetic trees. The P6 strain was predicted to contain three NRPS gene clusters, five type-I PKS gene clusters, and one type-I NRPS/PKS gene cluster via analysis at the antiSMASH Website. It may synthesize epichloenin A, fusaric acid, elsinochromes, and fusaridione A. Conclusions Based on its unique DNA sequence, taxonomic position in the phylogenetic tree and evolutional position, the yeast-like fungal strain P6 was identified as a novel species Aureobasidium hainanensis sp. nov. P6 isolate and had highly potential applications.

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.

Fructooligosaccharides (FOSs) are excellent food ingredients or feed additives by stimulating probiotics. In this paper, a CREA gene encoding a glucose repressor in the β-fructofuranosidase produce...

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.

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.

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.

Aureobasidium melanogenum HN6.2 is a high siderophore-producing yeast-like fungal strain. After blocking siderophore biosynthesis and attenuating the expression of the ornithine carbamoyltransferase gene (the OTC gene), the obtained D-LCFAO-cre strain produced 2.1 ± 0.02 mg of intracellular L-ornithine per mg of the protein. The overexpression of the L-ornithine decarboxylase gene (the SPE1-S gene) from Saccharomyces cerevisiae in the mutant D-LCFAO-cre could make the transformant E-SPE1-S synthesize 3.6 ± 0.1 of intracellular ornithine per mg of protein and produce 10.5 g/L of putrescine. The further overexpression of the ArgB/C gene encoding bifunctional acetylglutamate kinase/N-acetyl-gamma-glutamyl-phosphate reductase in the transformant E-SPE1-S caused the transformant E-SPE1-S-ArgB/C to accumulate L-ornithine (4.2 mg/mg protein) and to produce 21.3 g/L of putrescine. During fed-batch fermentation, the transformant E-SPE1-S-ArgB/C could produce 33.4 g/L of putrescine, the yield was 0.96 g/g of glucose, and the productivity was 0.28 g/L/h. The putrescine titer was much higher than that produced by most engineered strains obtained thus far.

Massoia lactone could be released from liamocins produced by Aureobasidium melanogenum M39. The obtained Massoia lactone was very stable and highly active against many fungal crop pathogens which cause many plant diseases and food unsafety. Massoia lactone treatment not only could effectively inhibit their hyphal growth and spore germination, but also caused pore formation in cell membrane, reduction of ergosterol content, rise in intracellular ROS levels, and leakage of intracellular components, consequently leading to cellular necrosis and cell death. The direct contact of Massoia lactone with Fusarium graminearum spores could stop the development of Fusarium head blight symptom in the diseased wheats. Therefore, Massoia lactone could be a promising candidate for development as an effective and green bio-fungicide because of its high anti-fungal activity and the multiplicity of mode of its action.

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.

The yeast Saccharomyces cerevisiae has been widely applied in fermentation industries, chemical industries and biological research and it is widespread in different environments, especially in sugar-rich environments. However, little is known about the occurrence, distribution and roles of S. cerevisiae in marine environments. In this study, only 10 strains among all the yeasts isolated from different marine environments belonged to S. cerevisiae. It was found that most of the strains of S. cerevisiae in marine environments occurred in guts, the surface of marine fish and mangrove trees. In contrast, they were not found in seawater and sediments. All the strains of S. cerevisiae isolated from the marine environments had a lower ability to produce ethanol than the highly alcohol-producing yeast Saccharomyces sp. W0 isolated from fermented rice, but the strains 2E00400, 2E00558, 2E00498, 2E00723, 2E00724 could produce higher concentrations of ethanol than any other marine-derived strains of S. cerevisiae obtained in this study. However, some of them had higher ethanol tolerance and higher trehalose content than Saccharomyces sp. W0. In particular, ethanol tolerance of the yeast strain 2E00498 was higher than that of Saccharomyces sp. W0. This may be related to the harsh marine environments from which they were isolated. Such yeast strains with higher alcohol tolerance could be used to further improve the alcohol tolerance of Saccharomyces sp. W0.

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.

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.

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.

Aureobasidium spp. can use a wide range of substrates and are widely distributed in different environments, suggesting that they can sense and response to various extracellular signals and be adapted to different environments. It is true that their pullulan, lipid and liamocin biosynthesis and cell growth are regulated by the cAMP-PKA signaling pathway; Polymalate (PMA) and pullulan biosynthesis is controlled by the Ca

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.

Isomaltulose is an ideal functional sweetener and has been approved as a safe sucrose substitute. It is produced mainly through sucrose isomerization catalyzed by sucrose isomerase. Here, to produce food-grade isomaltulose and improve its yield, the sucrose isomerase gene from Pantoea dispersa UQ68J was overexpressed in the non-pathogenic yeast Yarrowia lipolytica. When the engineered strain, S47, was fermented on 600 g/L sucrose in a 10-L bioreactor, a maximum isomaltulose concentration of 572.1 g/L was achieved. Sucrose isomerase activity was 7.43 U/mL, and yield reached 0.96 g/g. Moreover, monosaccharide byproducts were simultaneously transformed into intracellular lipids, thus reducing the production of undesirable compounds and resulting in high isomaltulose purity (97.8%) in the final broth. In summary, the bioprocess employed in this study provides an efficient alternative strategy for isomaltulose production.

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.

The detection of Helicobacter pylori infection in human feces is an appropriate non-invasive diagnostic method. However, the antibody-dependent stool antigen immunoassay bears many challenges. Therefore, we developed an antibody-independent biosensing platform. The core of this platform was a triple-module biosensor. The first module was Ca2+-doped superparamagnetic nanoparticles modified with an H. pylori-specific aptamer, functioning to selectively capture H. pylori cells from samples. The second module was a bifunctional co-polymer of chloroprotoporphyrin IX iron (III)-polyethylene glycol-desferrioxamine, which could bind to H. pylori with high affinity and chelate Fe3+ from the third module of Fe3+-quenched carbon dots (CDs) solution. When the formed module 1-H. pylori-module 2 complexes reacted with module 3, a subsequent magnetic separation could scavenge Fe3+, causing fluorescence recovery from quenched CDs as the transducing mechanism. This transducer could respond to tiny changes in Fe3+ concentration with distinguishable fluorescence differences, thus conferring the biosensor with high sensitivity, a wide detection range of 10–107 CFU/mL and a limit of detection (LOD) as low as 1 CFU/mL. From simulated human stool samples, H. pylori was enriched with a centrifugal microfluidic plate to eliminate any interference from matrices, and the bacteria were subjected to detection using the biosensor. The actual LOD for the biosensing platform coupling microfluidics and the biosensor was 101, and the total time taken was 65 min. This work showcases an instant, accurate, and ultra-sensitive diagnosis of H. pylori in feces.