Inbiose N.v.

Universiteit Gent

Puratos Nv

Puratos Naamloze Vennootschap

Joeri Beauprez

Jo Maertens

Marjan De Mey

Annelies Vercauteren

Pieter Coussement

Gaspard Lequeux

Gert Peters

Dries Van Herpe

Thomas Decoene

Filip Arnaut

Sofie De Maeseneire

Eric Vandamme

Tom Verhaeghe

Eric Timmermans

Nausicaä Lannoo

Sofie Aesaert

Sofie Parmentier

Kristof Vandewalle

This disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure is in the technical field of fermentation of metabolically engineered microorganisms. This disclosure describes engineered micro-organisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These micro-organisms can dephosphorylate N-acetylglucosamine-6-phosphate to N-acetyl glucosamine and convert the N-acetylglucosamine to N-acetylmannosamine. These micro-organisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, this disclosure provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein the microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetyl-glucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

In vivo synthesis of sialylated compounds

The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to newly identified sialyltransferases having alpha-2,3-sialyltransferase activity on the galactose residue of lactose. The invention also describes methods for the production of a 3'sialylated oligosaccharide using any one of the newly identified sialyltransferases as well as the purification of the 3′sialylated oligosaccharide. The present invention also provides a cell for production of the 3′sialylated oligosaccharide and the use of the cell in a cultivation or incubation.

Sialyltransferases for the production of sialylated oligosaccharides

The present invention relates to genetically engineered organisms, especially microorganisms such as bacteria and yeasts, for the production of added value bio-products such as specialty saccharide, activated saccharide, nucleoside, glycoside, glycolipid or glycoprotein. More specifically, the present invention relates to host cells that are metabolically engineered so that they can produce said valuable specialty products in large quantities and at a high rate by bypassing classical technical problems that occur in biocatalytical or fermentative production processes.

Metabolically engineered organisms for the production of added value bio-products

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a cell metabolically engineered for production of a mixture of at least four different neutral non-fucosylated oligosaccharides. Furthermore, the disclosure provides a method for the production of a mixture of at least four different neutral non-fucosylated oligosaccharides by a cell as well as the purification of at least one of the oligosaccharides from the cultivation.

Production of a mixture of neutral non-fucosylated oligosaccharides by a cell

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered microorganisms. The present invention describes engineered microorganisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These microorganisms can dephosphorylate N-acetylglucosamine-6-phosphate to N-acetylglucosamine and convert the N-acetylglucosamine to N-acetylmannosamine. These microorganisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetylglucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

The present invention relates to a method to produce mutated microorganisms which resist the phenomenon of lactose killing and to the microorganisms obtainable via said method. Such engineered microorganisms can be applied for the production of specialty products, such as but not limited to specialty carbohydrates, glycolipids and galactosylated compounds.

Mutant microorganisms resistant to lactose killing

The present invention relates to mutated and/or transformed microorganisms for the synthesis of various compounds. More specifically, the present invention discloses microorganisms mutated in the genes encoding for the regulators ArcA and IclR. The latter mutations result in a significant upregulation of the genes that are part of the colanic acid operon. Hence, said microorganisms are useful for the synthesis of any compound being part of the colanic acid pathway such as GDP-fucose, GDP-mannose and colanic acid, and/or, can be further used&#x2014;starting form GDP-fucose as a precursor&#x2014;to synthesize fucosylated oligosaccharides or&#x2014;starting from GDP-mannose as a precursor&#x2014;to synthesize mannosylated oligosaccharides. In addition, mutations in the genes coding for the transcriptional regulators ArcA and IclR lead to an acid resistance phenotype in the exponential growth phase allowing the synthesis of pH sensitive molecules or organic acids.

Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides

The present invention is related to a method for inhibiting nicotinamide coenzyme degradation in a cereal flour or wheat based product, comprising the addition of an effective amount of nucleotide pyrophosphatase inhibitor to said cereal flour based product, product such as a wheat based product. A further aspect of the present invention is a nucleotide pyrophosphatase having an amino acid N-terminal sequence being (G)IDDRHEVDLPPRP. In another aspect of the present invention, a dough comprising a nucleotide pyrophosphatase inhibitor such as pyrophosphate, and optionally a coenzyme regeneration system comprising at least one NAD(P) or NAD(P)H dependent hydrogenase or dehydrogenase is disclosed. Preferably the coenzyme regeneration system comprises (consists of) mannitol dehydrogenase and D-fructose.

Growing community of inventors

Bredene, Belgium

Joeri Beauprez