Patent ID: 8546105

Claim:
A modified eukaryotic cell capable of producing CMP-N-acetylneuraminate (CMP-Neu5Ac) above levels produced before said cell was modified, when provided N-acetylmannosamine (ManNAc), wherein said cell is modified to comprise and express: i) at least one copy of a cytidine monophosphate sialic acid synthetase (CMP-SAS) gene, operably-linked to a promoter functional in the eukaryotic cell, wherein said CMP-SAS gene encodes a polypeptide that catalyzes the conversion of N-acetylneuraminate (Neu5Ac) and cytidine triphosphate (CTP) to CMP-Neu5Ac and PPi; and wherein said polypeptide is (a) a polypeptide having at least 95% identity to SEQ ID NO:4; or (b) a polypeptide having at least 90% identity to SEQ ID NO:4 that contains conservative amino acid substitutions, in which CMP-SAS function is conserved; or (c) a variant of a polypeptide specified in (a) or (b) that contains one or more fusions or truncations, in which CMP-SAS function is conserved; and ii) at least one copy of a sialic acid phosphate synthetase (SAS) gene, operably-linked to a promoter functional in the eukaryotic cell, wherein said SAS gene encodes a polypeptide that catalyzes the conversion of N-acetylmannosamine 6-phosphate (ManNAc-6-P) and phosphoenolpyruvate (PEP) to N-acetylneuraminate-9-phosphate (NeuNAc-9-P) and Pi; and wherein said polypeptide is (d) a polypeptide having at least 95% identity to SEQ ID NO: 6; or (e) a polypeptide having at least 90% identity to SEQ ID NO:6 that contains conservative amino acid substitutions and in which SAS function is conserved; or (f) a variant of a polypeptide specified in (d) or (e) that contains one or more fusions or truncations, in which SAS function is conserved.