Guang-Lei Liu

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.

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.

Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11

The MIG1 gene of Saccharomycopsis fibuligera A11 was cloned from its genomic DNA using the degenerated primers and inverse PCR. The MIG1 gene (1152bp, accession number: HM450676) encoded a 384-amino acid protein very similar to Mig1s from other fungi. Besides their highly conserved zinc fingers, the Mig1 proteins displayed short conserved motifs of possible significance in glucose repression. The MIG1 gene in S. fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the MIG1 gene. The disruptant A11-c obtained could grow in the media containing hygromycin and 2-deoxy-d-glucose, respectively. α-Amylase, glucoamylse, acid protease and β-glucosidase production by the disruptant and expression of their genes in the disruptant were greatly enhanced. This confirms that Mig1, the transcriptional repressor, indeed regulates expression of the genes and production of the extracellular enzymes in S. fibuligera A11. At the same time, it was found that cell budding was enhanced and mycelial formation was reduced in the disruptant.

Marine yeasts as biocontrol agents and producers of bio-products.

As some species of marine yeasts can colonize intestine of marine animals, they can be used as probiotics. It has been reported that β-glucans from marine yeast cells can be utilized as immuno-stimulants in marine animals. Some siderophores or killer toxins produced by marine yeasts have ability to inhibit growth of pathogenic bacteria or kill pathogenic yeasts in marine animals. The virulent factors from marine pathogens can be genetically displayed on marine yeast cells, and the yeast cells displaying the virulent factors can stimulate marine animals to produce specific antibody against the pathogens. Some marine yeast cells are rich in proteins and essential amino acids and can be used in nutrition for marine animals. The marine yeast cells rich in lipid can be used for biodiesel production. Recently, it has been reported that some strains of Yarrowia lipolytica isolated from marine environments can produce nanoparticles. Because many marine yeasts can remove organic pollutants and heavy metals, they can be applied to remediation of marine environments. It has been shown that the enzymes produced by some marine yeasts have many unique properties and many potential applications.

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.

Taxonomy of Aureobasidium spp. and biosynthesis and regulation of their extracellular polymers

Abstract The genus Aureobasidium spp. have been divided into three species, A. pullulans. A. leucospermi and A. proteae, and A. pullulans has been known to have five varieties. However, after analysis of many strains of this yeast isolated from different environments, they do not belong to any of the three species or the five varieties. Although pullulan produced by A. pullulans has been widely used in different fields in industry and different strains of this yeast has been known to produce poly(β-L-malic acid) (PMA), heavy oils and β-1,3-glucan, it is still unknown how the black yeast synthesizes and secretes the extracellular polymers at molecular level. In this review article, new biosynthetic pathways of pullulan, PMA and heavy oils, the enzymes and their genes related to their biosynthesis and regulation are proposed. Furthermore, some enzymes and their genes related to pullulan biosynthesis in A. pullulans have been characterized. But it is completely unknown how pullulan is secreted and how PMA, heavy oils and β-1,3-glucan are synthesized and secreted. Therefore, there is much work to be done about taxonomy and biosynthesis, secretion and regulation of pullulan, PMA, heavy oils and β-1,3-glucan at molecular levels in Aureobasidium spp.

Yeast killer toxins, molecular mechanisms of their action and their applications

Abstract Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.

Chemical and biological characterization of siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 and its antibacterial activity

After analysis using HPLC and electronic ion spray mass spectroscopy, the purified siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 was found to be fusigen. The purified desferric fusigen still had strong inhibition of growth of the pathogenic Vibrio anguillarum while the fusigen chelated by Fe3+ lost the ability to inhibit the growth of the pathogenic bacterium. The added iron in the medium repressed expression of the hydroxylase gene encoding ornithine N5-oxygenase that catalyzes the N5-hydroxylation of ornithine for the first step of siderophore biosynthesis in the yeast cells while expression of the hydroxylase gene in the yeast cells grown in the medium plus ornithine was enhanced.