Zhen-Ming Chi

Biotechnological potential of inulin for bioprocesses.

Inulin consists of linear chains of β-2,1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. In this review article, inulin and its applications in bioprocesses are overviewed. The tubers of many plants, such as Jerusalem artichoke, chicory, dahlia, and yacon contain a large amount of inulin. Inulin can be actively hydrolyzed by microbial inulinases to produce fructose, glucose and inulooligosaccharides (IOS). The fructose and glucose formed can be further transformed into ethanol, single-cell protein, single cell oil and other useful products by different microorganisms. IOS formed have many functions. Therefore, inulin can be widely used in food, feed, pharmaceutical, chemical and biofuels industries.

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.

Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu

Trehalose accumulation from starch by Saccharomycopsis fibuligera sdu was examined in 300-ml shaken flask culture and Biostat B(2) 2-1 fermentation. In the 300-ml flask, 16.5% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed with 100-ml medium shaken at 200 rpm for 50 h at 30 degrees C. We found that 1.0% soluble starch in the medium was most suitable for trehalose accumulation by this yeast strain. In the Biostat B(2) 2-1 fermentor, 18.0% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed within 48 h of fermentation when agitation speed was 200 rpm. The trehalose obtained from the yeast cells was identical to standard trehalose from Sigma based on the analysis results of High-Performance Exchange Anionic Chromatography (HPEAC).

Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68.

Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.

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.

Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containingmaterials

Yarrowia lipolytica ACA-DC 50109 has been reported to be an oleaginous yeast and significant quantities of lipids were accumulated inside the yeast cells. In this study, the INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. The activity of the inulinase with 6 × His tag secreted by the transformant Z31 obtained was found to be 41.7U mL(-1) after cell growth for 78 h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g L(-1) within 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g L(-1) within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g L(-1) within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C(16:0), C(18:1) and C(18:2), especially C(18:1) (58.5%).

Role of phosphatidylinositol (PI) in ethanol production and ethanol tolerance by a high ethanol producing yeast

The effect of inositol addition on phospholipids, cell growth, ethanol production and ethanol tolerance in a high ethanol producing Saccharomyces sp were studied. Addition of inositol greatly influenced major phospholipid synthesis. With inositol in the fermentation medium, phosphatidylinositol (PI) content was increased, while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were decreased. However, without inositol in the fermentation medium, PI content dropped down within 24 h, then increased, but was lower than in the presence of inositol. When yeast cells had a higher content of PI, they produced ethanol much more rapidly and tolerated higher concentrations of ethanol. During ethanol shock treatment at 18% (v/v) ethanol, yeast cells with a higher concentration of PI lost their viability much more slowly than those with a lower concentration of PI, indicating that the PI content in these yeast cells can play an important role in ethanol production and ethanol tolerance. Fatty acids and ergosterol were not responsible for high ethanol tolerance and high ethanol production in this yeast strain.

Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes

Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish and marine algae were obtained. After lipase activity of the yeast cultures was estimated, we found that nine yeast strains obtained in this study grown in the medium with olive oil could produce lipase. The results of routine identification and molecular methods show that they belonged toCandida intermedia YA01a,Pichia guilliermondii N12c,Candida parapsilosis 3eA2,Lodderomyces elongisporus YF12c,Candida quercitrusa JHSb,Candia rugosa wl8,Yarrowia lipolytica N9a,Rhodotorula mucilaginosa L10-2 andAureobasidium pullulans HN2.3, respectively. The optimal pHs and temperatures of lipases produced by them were between 6.0 and 8.5 and between 35 and 40°C, respectively. Majority of lipases from the yeast strains were cell-bound and only lipase fromA. pullulans HN2.3 was extracellular. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry.