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Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast

It has been well documented that Aureobasidium pullulans is widely distributed in different environments. Different strains of A. pullulans can produce amylase, proteinase, lipase, cellulase, xylanase, mannanase, transferases, pullulan, siderophore, and single-cell protein, and the genes encoding proteinase, lipase, cellulase, xylanase, and siderophore have been cloned and characterized. Therefore, like Aspergillus spp., it is a biotechnologically important yeast that can be used in different fields. So it is very important to sequence the whole genomic DNA of the yeast cells in order to find new more bioproducts and novel genes from this yeast.

Saccharomycopsis fibuligera and its applications in biotechnology.

Saccharomycopsis fibuligera is found to actively accumulate trehalose from starch and the gene responsible for biosynthesis of trehalose has been cloned and its expression has been characterized. This yeast is also found to secrete a large amount of amylases, acid protease and beta-glucosidase which have highly potential applications in fermentation industry. The genes encoding amylases, acid protease and beta-glucosidase in S. fibuligera have been cloned and characterized. It is also used to produce ethanol from starch, especially cassava starch by co-cultures of Saccharomyces cereviase or Zymomonas mobilis.

Bioethanol production from hydrolysates of inulin and the tuber meal of Jerusalem artichoke by Saccharomyces sp. W0.

It has been confirmed that Saccharomyces sp. W0 can produce high concentration of ethanol. However, this yeast strain cannot secrete inulinase. Therefore, in this study, inulin was hydrolyzed into reducing sugar by the recombinant inulinase produced by Pichia pastoris X-33/pPICZaA-INU1. It was found that 38.2U of the recombinant inulinase per gram of inulin was suitable for the inulin hydrolysis and ethanol production by Saccharomyces sp. W0 and the fermentation period was 120 h. At the end of the fermentation, over 14.6 ml of ethanol per 100ml of the fermented medium was produced, the ethanol productivity was over 0.384 g of ethanol/g of inulin and over 98.8% of total sugar was utilized. When the Saccharomyces sp. W0 was grown in the mixture of 4.0% hydrolysate of soybean meal and 20.0% of the hydrolysate of inulin for 120 h, over 14.9 ml of ethanol per 100ml of the fermented medium was yielded, the ethanol productivity was over 0.393 g of ethanol/g of inulin and 98.9% of total sugar was used by the yeast strain. When Saccharomyces sp. W0 carrying the same inulinase gene was grown in the medium containing 50 g of the tuber meal of Jerusalem artichoke per 100ml for 144 h, over 12.1+/-0.35%ml of ethanol per 100ml of the fermented medium was yielded, the ethanol productivity was 0.319+/-0.9 g of ethanol/g of sugar and 3.7% (w/v) of total sugar and 0.5% (w/v) of reducing sugar were left in the fermented media.

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 x His tag was found to be 22.6 U mg(-1) of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 degrees C, respectively and the inulinase was stable in the pH range of 3-8 and in the temperature range of 0-50 degrees C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g(-1) of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L(-1) citric acid and 5.3 g L(-1) iso-citric acid from inulin while 68.9 g L(-1) of citric acid and 4.1 g L(-1) iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.

Production, characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.

Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109.

In this study, the MIG1 gene in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109 (the parent yeast) was disrupted and the disruptant M25 obtained could grow in yeast nitrogen base-N5000 medium without uracil or the medium with 2-deoxy-D-glucose. It was found that the cells of the disruptant M25 had more lipid bodies than those of its parent yeast. The disruptant M25 contained 48.7% (w/w) of oil based on its cell weight while the parent yeast only contained 36.0% (w/w) of oil. Transcript levels of many genes relevant to lipid biosynthesis in the disruptant M25 were enhanced compared to those of the same genes in the parent yeast. However, transcript level of the MFE1 gene, one of the genes relevant to fatty acid degradation was reduced in the disruptant M25 compared to that of the same gene in the parent yeast. Such changes in gene expression profile may cause the increased lipid biosynthesis in the disruptant M25. Biosynthesis of C18:1 fatty acid in the disruptant M25 was greatly enhanced compared to that in the parent yeast.

Siderophore production by the marine-derived Aureobasidium pullulans and its antimicrobial activity

Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, only the yeast strain HN6.2 which was identified to be Aureobasidium pullulans was found to produce high level of the siderophore. Under the optimal conditions, this yeast strain could produce 1.1mg/ml of the siderophore. The crude siderophore produced by the yeast strain HN6.2 was able to inhibit cell growth of Vibrio anguillarum and Vibrio parahaemolyticus, isolated from the diseased marine animals.

High level lipid production by a novel inulinase-producing yeast Pichia guilliermondii Pcla22.

In this study, an inulinase-producing yeast strain Pcla22 of Pichia guilliermondii was identified. It was found that the yeast strain Pcla22 could produce higher amount of oil and more lipid bodies in its cells than any other yeast strains tested in this study. Under the optimal conditions, 60.6%(w/w) of lipid based on cell dry weight, 20.4 g/l of the dry cell mass, SCO produced per g of consumed sugar of 0.19 g/g and biomass produced per g of consumed sugar of 0.32 g/g were obtained in the culture of the yeast strain Pcla22 after 96 h of the fed-batch fermentation. Over 79.8% of the fatty acids from the yeast strain Pcla22 grown in the oil production medium containing inulin was C(16:0) and C(18:1), especially C(18:1) (57.9%). The biodiesel obtained from the produced lipid could be burnt well.

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C(16:0), C(18:1) and C(18:2), especially C(18:1) (50.6%).

Molecular characterization and expression of microbial inulinase genes.

Many genes encoding exo- and endo-inulinases from bacteria, yeasts and filamentous fungi have been cloned and characterized. All the inulinases have several conserved motifs, such as WMND(E)PNGL, RDP, EC(V)P, SVEVF, Q and FS(T), which play an important role in inulinase catalysis and substrate binding. However, the exo-inulinases produced by yeasts has no conserved motif SVEVF and the yeasts do not produce any endo-inulinase. Exo- and endo-inulinases found in different microorganisms cluster separately at distant positions from each other. Most of the cloned inulinase genes have been expressed in Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Klyuveromyces lactis and Escherichia coli, respectively. The recombinant inulinases produced and the engineered hosts using the cloned inulinase genes have many potential applications. Expression of most of the inulinase genes is repressed by glucose and fructose and induced by inulin and sucrose. However, the detailed mechanisms of the repression and induction are still unknown.