This disclosure provides epimerase enzymes useful for commercial scale production of allulose from fructose. The disclosed enzymes (“epimerase variants”) are variants of Burkholderia multivorans CGD1 xylose isomerase engineered to have improved catalytic activity of about 1.5- to 2-fold compared with the parent enzyme.

Disclosed is a method for promoting extracellular expression of proteins in B. subtilis using cutinase, which belongs to the technical fields of genetic engineering, enzyme engineering and microbial engineering. It teaches co-expressing a cutinase mutant and a target protein in B. subtilis to promote extracellular expression of the target protein which is naturally located inside cells. The target protein includes xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, branching enzyme and the like. The invention can achieve extracellular expression of intracellularly localized target protein, improve the production efficiency, reduce the production cost and simplify the subsequent extraction process.

Disclosed is a method for promoting extracellular expression of proteins in B. subtilis using cutinase, which belongs to the technical fields of genetic engineering, enzyme engineering and microbial engineering. It teaches co-expressing a cutinase mutant and a target protein in B. subtilis to promote extracellular expression of the target protein which is naturally located inside cells. The target protein includes xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, branching enzyme and the like. The invention can achieve extracellular expression of intracellularly localized target protein, improve the production efficiency, reduce the production cost and simplify the subsequent extraction process.

The present invention relates to a yeast cell that is genetically modified comprising: a) a disruption of one or more aldehyde dehydrogenase (E.C:1.2.1.4) native to the yeast; b) one or more nucleotide sequence encoding a heterologous NAD.sub.+-dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10); c) one or more nucleotide sequence encoding a homologous or heterologous acetyl-CoA synthetase (E.C. 6.2.1.1); and d) a modification that leads to reduction of glycerol 3-phosphate phosphohydrolase (E.C. 3.1.3.21) and/or glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8 or E.C. 1.1.5.3) activity, native to the yeast.

The present invention relates to a yeast cell that is genetically modified comprising: a) a disruption of one or more aldehyde dehydrogenase (E.C:1.2.1.4) native to the yeast; b) one or more nucleotide sequence encoding a heterologous NAD.sub.+-dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10); c) one or more nucleotide sequence encoding a homologous or heterologous acetyl-CoA synthetase (E.C. 6.2.1.1); and d) a modification that leads to reduction of glycerol 3-phosphate phosphohydrolase (E.C. 3.1.3.21) and/or glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8 or E.C. 1.1.5.3) activity, native to the yeast.

The present specification relates to a transformed yeast strain capable of simultaneously utilizing xylose and glucose as carbon sources, a preparation method thereof and a biofuel production method using the same. The transformed yeast strain transforms a wild-type yeast strain incapable of using xylose as a carbon source and simultaneously convert glucose and xylose, thereby enabling high yield production of a biofuel. The economics and sustainability of the biofuel and biomaterial production processes can be highly enhanced by providing a strain which can easily be converted to a strain capable of producing a biofuel/material in a high yield through an additional modification.

The present specification relates to a transformed yeast strain capable of simultaneously utilizing xylose and glucose as carbon sources, a preparation method thereof and a biofuel production method using the same. The transformed yeast strain transforms a wild-type yeast strain incapable of using xylose as a carbon source and simultaneously convert glucose and xylose, thereby enabling high yield production of a biofuel. The economics and sustainability of the biofuel and biomaterial production processes can be highly enhanced by providing a strain which can easily be converted to a strain capable of producing a biofuel/material in a high yield through an additional modification.

Acetate is a potent microbial inhibitor which can affect the performance of yeast in ethanolic fermentation. The present disclosure provides a recombinant microbial host cell having (i) a first genetic modification for increasing the activity of one or more proteins that function in a first metabolic pathway to convert acetate into an alcohol in the microbial host cell; (ii) a second genetic modification for increasing the activity of one or more proteins that function in a second metabolic pathway to import glycerol in the recombinant microbial host cell (iii) a third genetic modification for increasing the activity of one or more proteins that function in a third metabolic pathway to convert a C5 carbohydrate into ethanol in the microbial host cell. The recombinant microbial host cell comprises and natively expresses native proteins that function in a fourth native metabolic pathway to produce glycerol in the microbial host cell.

Acetate is a potent microbial inhibitor which can affect the performance of yeast in ethanolic fermentation. The present disclosure provides a recombinant microbial host cell having (i) a first genetic modification for increasing the activity of one or more proteins that function in a first metabolic pathway to convert acetate into an alcohol in the microbial host cell; (ii) a second genetic modification for increasing the activity of one or more proteins that function in a second metabolic pathway to import glycerol in the recombinant microbial host cell (iii) a third genetic modification for increasing the activity of one or more proteins that function in a third metabolic pathway to convert a C5 carbohydrate into ethanol in the microbial host cell. The recombinant microbial host cell comprises and natively expresses native proteins that function in a fourth native metabolic pathway to produce glycerol in the microbial host cell.

Disclosed herein are improved processes for making mannose including the step of converting the M6P to mannose, catalyzed by a M6PP, using enzymes with higher activities compared to M6PPs previously used in a process to produce mannose.