Polysaccharide compositions and methods of producing, screening, and formulating polysaccharide compositions

Abstract

Provided herein are polysaccharide compositions and methods of culturing microalgae to produce polysaccharides. Also provided are methods of using polysaccharides for applications such as reducing cholesterol in mammals, inactivating viruses, stabilizing foods, and other uses. Also provided are transgenic algae capable of utilizing fixed carbon sources for energy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows precipitation of 4 liters of Porphyridium cruentum exopolysaccharide using 38.5% isopropanol. (a) supernatant; (b) addition of 38.5% isopropanol; (c) precipitated polysaccharide; (d) separating step.

FIG. 2 shows Porphyridium sp. cultured on agar plates containing various concentrations of zeocin.

FIG. 3 shows growth of Porphyridium sp. and Porphyridium cruentum cells grown in light in the presence of various concentrations of glycerol.

FIG. 4 shows Porphyridium sp. cells grown in the dark in the presence of various concentrations of glycerol.

FIG. 5 shows levels of solvent-accessible polysaccharide in Porphyridium sp. homogenates subjected to various amounts of physical disruption from Sonication Experiment 1.

FIG. 6 shows levels of solvent-accessible polysaccharide in Porphyridium sp. homogenates subjected to various amounts of physical disruption from Sonication Experiment 2.

FIG. 7 shows sexually transmitted disease prevention devices containing various amounts of exopolysaccharide.

FIG. 8 shows protein concentration measurements of autoclaved, protease-treated, and diafiltered exopolysaccharide.

FIG. 9 shows various amounts and ranges of amounts of compounds found per gram of cells in cells of the genus Porphyridium.

Claims

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24. A polysaccharide produced from a cell of the genus Porphyridium, comprising xylose, glucose, and galactose wherein the molar amount of glucose in the polysaccharide is at least 65% of the molar amount of galactose.

25. The polysaccharide of claim 24, wherein the molar amount of glucose in the polysaccharide is at least 75% of the molar amount of galactose.

26. The polysaccharide of claim 24, wherein the molar amount of glucose in the polysaccharide is greater than the molar amount of galactose.

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122. A method of producing a polysaccharide comprising culturing a microalgae cell in the presence of at least 0.01 micromolar of a compound, wherein the compound is incorporated into the polysaccharide produced by the cell.

123. The method of claim 122, wherein the compound is selected from Tables 2 or 3.

124. The method of claim 122 wherein the cells are selected from the species listed in Table 1.

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128. A cell of the genus Porphyridum comprising an exogenous gene that encodes a carbohydrate transporter protein.

129. The cell of claim 128, wherein the protein has at least 60% amino acid sequence identity with a protein selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26 and 27.

130. The cell of claim 128, wherein the cell contains a nucleic acid that has at least 60% nucleotide identity with a nucleic acid selected from the group consisting of SEQ ID NOs: 21, 23 and 25.

131. A method of trophically converting a cell of the genus Porphyridium comprising: a. providing a nucleic acid encoding a carbohydrate transporter protein;b. transforming the cell with the nucleic acid; andc. selecting for the ability to undergo cell division i. in the absence of light; andii. in the presence of a carbohydrate that is transported by the carbohydrate transporter protein.

132. The method of claim 131, wherein the carbohydrate transporter protein has at least 60% amino acid identity with one or more of SEQ ID NOs: 20, 22, 24, 26 and 27.

133. The method of claim 131, wherein the nucleic acid encoding a carbohydrate transporter protein is in operable linkage with a promoter active in microalgae.

134. The method of claim 131, wherein the carbohydrate is selected from Tables 2 or 3.

135. An expression vector comprising a nucleic acid sequence encoding a carbohydrate transporter protein, wherein the nucleic acid has at least 60% nucleotide identity with one or more of SEQ ID NOs: 21, 23 or 25.

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166. A method of producing a glycopolymer comprising a. providing a transgenic cell containing a recombinant gene encoding a monosaccharide transporter; andb. culturing the cell in the presence of at least one monosaccharide, wherein the monosaccharide is transported by the transporter into the cell and is incorporated into the glycopolymer.

167. The method of claim 166, wherein the glycopolymer is a polysaccharide.

168. The method of claim 166, wherein the cell is a microalgae.

169. The method of claim 168, wherein the cell is selected from Table 1.

170. The method of claim 169, wherein the cell is of the genus Porphyridium.

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172. The method of claim 166, wherein the polysaccharide is enriched for the at least one monosaccharide compared to an endogenous polysaccharide produced by a non-transgenic cell of the same species.

173. The method of claim 172, wherein the monosaccharide is selected from the group consisting of arabinose, fructose, fucose, galactose, glucose, mannose, xylose, glucuronic acid, glucosamine, galactosamine, rhamnose and N-acetyl glucosamine.

174. The method of claim 170, wherein the transporter has a lower Km for glucose than at least one monosaccharide selected from the group consisting of galactose, xylose, glucuronic acid, mannose, and rhamnose.

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177. The method of claim 170, wherein the transporter has a lower Km for glucuronic acid than at least one monosaccharide selected from the group consisting of glucose, galactose, xylose, mannose, and rhamnose.

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180. The method of claim 170, wherein the cell is cultured in the presence of at least two monosaccharides, both of which are transporter by the transporter.

181. The method of claim 180, wherein the two monosaccharides are selected from the list consisting of glucose, galactose, xylose, glucuronic acid, rhamnose and mannose.

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