The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence anal...The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.展开更多
Fungal strain PA-2 was isolated from infected poplar leaves from the Ping’an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer(ITS) sequence o...Fungal strain PA-2 was isolated from infected poplar leaves from the Ping’an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer(ITS) sequence of its 16S r DNA, the strain was identified as Aureobasidium pullulans. The culture and metabolites of strain PA-2 showed high herbicidal potential to five tested weeds Galium aparine var. tenerum, Chenopodium album, Malva crispa, Polygonum lapathifolium and Avena fatua. For the in vitro test, 5 days after the detached leaves were inoculated with PA-2 culture, all leaves infected by the hyphae and became black and rotten. For the in vivo test, a metabolite filtrate of PA-2 culture was sprayed over the living weed plants, and five days after inoculation, the weed plants became withered and necrotic. Seven days after inoculation, the fresh weight reductions of G. aparine var. tenerum, C. album, M. crispa, P. lapathifolium and A. fatua were 87.25, 78.46, 82.25, 62.11, and 80.27%, respectively. Galium aparine var. tenerum and M. crispa exhibited significant reductions in fresh weight. The bio-safety test on the five crops showed no significant plant height reductions, which was also observed for wheat(Triticum aestivum), faba bean(Vicia faba), and barley(Hordeum vulgare). By contrast, oilseed rape(Brassica napus) and pea(Pisum sativum) exhibited light spots but no significant reductions in plant height. These results indicated that A. pullulans could be a potential microbial herbicide for the control of the target weeds in crops. Optimization of the carbon and nitrogen sources for cultural media and substances for solid-state fermentation indicated that PA-2 had better colony growth and spore production with the optimal carbon source of glucose(C4), nitrogen source of soybean flour(N2), and the optimal substance was wheat bran. The results in this study provide useful information for the development ofA. pulluans PA-2 as an herbicide for bio-control of the weed.展开更多
We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maxi...We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U(mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 k Da. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other fungi. The optimal p H of the enzyme was 5.6, and the activity profile was stable in a range of acidity(p H 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. Km and Vmax values of the purified enzyme were 4.7 mg m L-1 and 0.57 μmol L-1 min-1(mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose(CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp(EU978473). The protein deduced contained the conserved domain of cellulase superfamily(glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.展开更多
Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nin...Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureo-basidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.展开更多
In the current study, in order to change the permeability of cell membrane and solve the problem of linked group of fungi mycelium, the method of adjusting osmotic pressure of medium and adding tween-80 was establishe...In the current study, in order to change the permeability of cell membrane and solve the problem of linked group of fungi mycelium, the method of adjusting osmotic pressure of medium and adding tween-80 was established. The utilized strain with relatively high exopolysaccharide (EPS) yield and low pigment level was obtained after the rejuvenation and sifting of long-preserved Aureobasidium pullulans strain. The optimal proportion of substrate was determined by means of orthogonal test. The transformation ratio of EPS was increased by 10% - 20% and the pigment content was greatly reduced. The fermenting liquor is between creamy white and pale yellow, and the white primary product can be gained without decolourization step. Furthermore, to magnify to 5 L bioreactor can get the similar result.展开更多
Aureobasidium pullulans, a biocontrol agent for the annual weed Galium aparine L. was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X(recommended field rate), 0.5X, 0.2X, ...Aureobasidium pullulans, a biocontrol agent for the annual weed Galium aparine L. was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X(recommended field rate), 0.5X, 0.2X, 0.1X, 0.067 X, and 0.05 X concentrations. Germination of A. pullulans with paraquat, 2, 4-D, quizalofop-p, and clethodim treatment appeared reduced compared with germination of A. pullulans with fluroxypyr treatment at all concentrations. Stunted and shorter germ tubes in comparison with the control were observed with 2, 4-D, quizalofop-p, and clethodim at 0.2X. All concentration of paraquat, 2, 4-D, quizalofop-p, and clethodim except0.05 X, significantly decreased radial growth of A. pullulans compared with its growth on the untreated PDA medium. Field trials to further develop A. pullulans as biocontrol agent for control G. aparine L. was conducted to test the effectiveness of this fungus in wheat plots for 2 years at the same location in Xining. Treatments included spore suspensions of A. pullulans alone, a mixture of both fungus and fluroxypyr in wheat. Biocontrol agent effectiveness was estimated at approximately 7and 14 days after treatment, as disease incidence, percent weed control, and weed biomass reduction. Significant reduction in weed biomass occurred in combination treatments, and potential exists to tank mix A. pullulans with fluroxypyr. Leaf surface moisture and air temperatures following application may account for inconsistencies in field results between years. This fungal organisms show potential as bioherbicides for weeds in G. aparine L.展开更多
The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming.In this study,the engineered strains of A.pullulans var.aubasidani were found to be suitable for green fu...The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming.In this study,the engineered strains of A.pullulans var.aubasidani were found to be suitable for green fumaric acid producer.Removal and complementation of the relevant genes showed only the ornithine-urea cycle(OUC)was involved in high level fumarate biosynthesis which was controlled by the Ca^(2+)signaling pathway.Removal of both the GOX gene encoding glucose oxidase and the PKS1 gene encoding the polyketide synthase for 3,5-dihydroxydecanoic acid biosynthesis and overexpression of the PYC gene encoding pyruvate carboxylase made the strain e-PYC produce 88.1±4.3 g/L of fumarate at flask level and 93.9±0.8 g/L of fumarate during the fed-batch fermentation.As a yeast-like fungal strain,it was very easy to cultivate A.pullulans var.aubasidani DH177 and their mutants in the bioreactor and to edit its genomic DNAs to enhance fumarate production.It was found that 2 mol of CO_(2) could be fixed during a maximal theoretical yield of 2 mol of fumarate per mole of glucose consumed in the OUC.Therefore,the OUC-mediated fumarate biosynthesis pathway in A.pullulans var.aubasidani was a green and eco-friendly process for the global sustainable development and carbon neutrality.展开更多
β-Poly(L-malic acid)(PMLA)is a water-soluble biopolymer used in food,medicine and other industries.To date,the biosynthesis pathway of PMLA has not been fully elucidated.In this study,we sequenced the transcriptom e ...β-Poly(L-malic acid)(PMLA)is a water-soluble biopolymer used in food,medicine and other industries.To date,the biosynthesis pathway of PMLA has not been fully elucidated.In this study,we sequenced the transcriptom e of strain Aureobasidium melanogenum under 20 g/L CaCO_(3) addition.The resulting sequencing reads were assembled and annotated for the differentially expressed genes(DEGs)analysis and novel transcripts identification.The result indicated that with the CaCO_(3) addition,the tricarboxylic cycle(TCA)cycle and glyoxylate pathway were up-regulated,and it also found that a non-ribosomal peptide synthetase(NRPS)like protein was highly expressed.The DEGs analysis showed a high expression level of malate dehydrogenase(MDHC)and phosphoenolpyruvate carboxykinase(PCKA)in the CaCO_(3) group,which indicated a cytosolic malate activity.We speculated that the malate should be transported to or synthesized in the cytoplasm,which was then polymerized to PMLA by the NRPS-like protein,accompanied by the up-regulated TCA cycle providing ATP for the polymerization.Depending on the analysis,we assumed that an NRPS-like protein,the TCA cycle,and the cytosolic malate together are contributing to the PMLA biosynthesis.展开更多
文摘The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.
基金supported by the National Natural Science Foundation of China (31760539)the Natural Science Foundation of Qinghai Province, China (2018-ZJ-917)
文摘Fungal strain PA-2 was isolated from infected poplar leaves from the Ping’an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer(ITS) sequence of its 16S r DNA, the strain was identified as Aureobasidium pullulans. The culture and metabolites of strain PA-2 showed high herbicidal potential to five tested weeds Galium aparine var. tenerum, Chenopodium album, Malva crispa, Polygonum lapathifolium and Avena fatua. For the in vitro test, 5 days after the detached leaves were inoculated with PA-2 culture, all leaves infected by the hyphae and became black and rotten. For the in vivo test, a metabolite filtrate of PA-2 culture was sprayed over the living weed plants, and five days after inoculation, the weed plants became withered and necrotic. Seven days after inoculation, the fresh weight reductions of G. aparine var. tenerum, C. album, M. crispa, P. lapathifolium and A. fatua were 87.25, 78.46, 82.25, 62.11, and 80.27%, respectively. Galium aparine var. tenerum and M. crispa exhibited significant reductions in fresh weight. The bio-safety test on the five crops showed no significant plant height reductions, which was also observed for wheat(Triticum aestivum), faba bean(Vicia faba), and barley(Hordeum vulgare). By contrast, oilseed rape(Brassica napus) and pea(Pisum sativum) exhibited light spots but no significant reductions in plant height. These results indicated that A. pullulans could be a potential microbial herbicide for the control of the target weeds in crops. Optimization of the carbon and nitrogen sources for cultural media and substances for solid-state fermentation indicated that PA-2 had better colony growth and spore production with the optimal carbon source of glucose(C4), nitrogen source of soybean flour(N2), and the optimal substance was wheat bran. The results in this study provide useful information for the development ofA. pulluans PA-2 as an herbicide for bio-control of the weed.
基金Qingdao Municipal Science and Technology Commission,Qingdao,China for providing financial support to this work(06-2-2-22-jch)
文摘We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U(mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 k Da. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other fungi. The optimal p H of the enzyme was 5.6, and the activity profile was stable in a range of acidity(p H 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. Km and Vmax values of the purified enzyme were 4.7 mg m L-1 and 0.57 μmol L-1 min-1(mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose(CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp(EU978473). The protein deduced contained the conserved domain of cellulase superfamily(glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.
基金supported by grant No. 30771645 from the National Natural Science Foundation of China
文摘Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureo-basidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.
文摘In the current study, in order to change the permeability of cell membrane and solve the problem of linked group of fungi mycelium, the method of adjusting osmotic pressure of medium and adding tween-80 was established. The utilized strain with relatively high exopolysaccharide (EPS) yield and low pigment level was obtained after the rejuvenation and sifting of long-preserved Aureobasidium pullulans strain. The optimal proportion of substrate was determined by means of orthogonal test. The transformation ratio of EPS was increased by 10% - 20% and the pigment content was greatly reduced. The fermenting liquor is between creamy white and pale yellow, and the white primary product can be gained without decolourization step. Furthermore, to magnify to 5 L bioreactor can get the similar result.
基金Supported by National Natural Science Foundation of China(No.31160371,30860165)the National Key Technology R&D program of China(No.2012BAD19B02)the National High Technology Research and Development Program(863Program)of China(No.2011AA10A206)
文摘Aureobasidium pullulans, a biocontrol agent for the annual weed Galium aparine L. was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X(recommended field rate), 0.5X, 0.2X, 0.1X, 0.067 X, and 0.05 X concentrations. Germination of A. pullulans with paraquat, 2, 4-D, quizalofop-p, and clethodim treatment appeared reduced compared with germination of A. pullulans with fluroxypyr treatment at all concentrations. Stunted and shorter germ tubes in comparison with the control were observed with 2, 4-D, quizalofop-p, and clethodim at 0.2X. All concentration of paraquat, 2, 4-D, quizalofop-p, and clethodim except0.05 X, significantly decreased radial growth of A. pullulans compared with its growth on the untreated PDA medium. Field trials to further develop A. pullulans as biocontrol agent for control G. aparine L. was conducted to test the effectiveness of this fungus in wheat plots for 2 years at the same location in Xining. Treatments included spore suspensions of A. pullulans alone, a mixture of both fungus and fluroxypyr in wheat. Biocontrol agent effectiveness was estimated at approximately 7and 14 days after treatment, as disease incidence, percent weed control, and weed biomass reduction. Significant reduction in weed biomass occurred in combination treatments, and potential exists to tank mix A. pullulans with fluroxypyr. Leaf surface moisture and air temperatures following application may account for inconsistencies in field results between years. This fungal organisms show potential as bioherbicides for weeds in G. aparine L.
基金supported by Grant 31970058 from National Natural Science Foundation of China.
文摘The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming.In this study,the engineered strains of A.pullulans var.aubasidani were found to be suitable for green fumaric acid producer.Removal and complementation of the relevant genes showed only the ornithine-urea cycle(OUC)was involved in high level fumarate biosynthesis which was controlled by the Ca^(2+)signaling pathway.Removal of both the GOX gene encoding glucose oxidase and the PKS1 gene encoding the polyketide synthase for 3,5-dihydroxydecanoic acid biosynthesis and overexpression of the PYC gene encoding pyruvate carboxylase made the strain e-PYC produce 88.1±4.3 g/L of fumarate at flask level and 93.9±0.8 g/L of fumarate during the fed-batch fermentation.As a yeast-like fungal strain,it was very easy to cultivate A.pullulans var.aubasidani DH177 and their mutants in the bioreactor and to edit its genomic DNAs to enhance fumarate production.It was found that 2 mol of CO_(2) could be fixed during a maximal theoretical yield of 2 mol of fumarate per mole of glucose consumed in the OUC.Therefore,the OUC-mediated fumarate biosynthesis pathway in A.pullulans var.aubasidani was a green and eco-friendly process for the global sustainable development and carbon neutrality.
基金the financial support of the Tianjin Municipal Science and Technology Commission(17PTGCCX00190,17PTSYJC00080,17YFCZZC00310,and 16YFXTSF00460)the Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control(ZXKF20180301).
文摘β-Poly(L-malic acid)(PMLA)is a water-soluble biopolymer used in food,medicine and other industries.To date,the biosynthesis pathway of PMLA has not been fully elucidated.In this study,we sequenced the transcriptom e of strain Aureobasidium melanogenum under 20 g/L CaCO_(3) addition.The resulting sequencing reads were assembled and annotated for the differentially expressed genes(DEGs)analysis and novel transcripts identification.The result indicated that with the CaCO_(3) addition,the tricarboxylic cycle(TCA)cycle and glyoxylate pathway were up-regulated,and it also found that a non-ribosomal peptide synthetase(NRPS)like protein was highly expressed.The DEGs analysis showed a high expression level of malate dehydrogenase(MDHC)and phosphoenolpyruvate carboxykinase(PCKA)in the CaCO_(3) group,which indicated a cytosolic malate activity.We speculated that the malate should be transported to or synthesized in the cytoplasm,which was then polymerized to PMLA by the NRPS-like protein,accompanied by the up-regulated TCA cycle providing ATP for the polymerization.Depending on the analysis,we assumed that an NRPS-like protein,the TCA cycle,and the cytosolic malate together are contributing to the PMLA biosynthesis.
