US 11,535,878 B2 · In vivo synthesis of sialylated compounds

Abstract

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.

Claims12

Dependent claims

A method for the production of a sialylated compound in a microorganism, the method consisting essentially of:

culturing a microorganism in a culture medium, said culture medium comprising an exogenous precursor, wherein said microorganism comprises at least one nucleic acid encoding a phosphatase, at least one nucleic acid encoding an N-acetylmannosamine epimerase;
and at least one nucleic acid encoding a sialic acid synthase, and wherein said microorganism is unable to i) convert N-acetylglucosamine-6-P to glucosamine-6-P, ii) convert N-acetyl-glucosamine to N-acetyl-glucosamine-6-P, and iii) convert N-acetyl-neuraminate to N-acetyl-mannosamine;
and increasing expression in said microorganism of a nucleic acid encoding a HAD-alike phosphatase, wherein said HAD-alike phosphatase comprises:
at least one of the following motifs:
Motif 1:
hDxDx[TV] (SEQ ID NO:
73], or Motif 2:
[GSTDE] [DSEN]x(1-2)[hP]x(1-2)[DGTS] (SEQ ID NOs:
74, 75, 76, 77) wherein h means a hydrophobic amino acid (A, I, L, M, F, V, P, G) and x can be any distinct amino acid;
or any one of SEQ ID NOs:
43, 44, 45, 46, 47, 48, 50, 51, 52, 54, 55, or 57.

8 more elements

Dependencies are inferred from the claim text.

In vivo synthesis of sialylated compounds

Life of this patent

In vivo synthesis of sialylated compounds

Life of this patent

Abstract

Claims12