US20110165626A1 – High Yield Production of Sialic Acid (Neu5ac) by Fermentation.

[evelynontiveros]

<br> “Commercial scale” refers to gram scale production of a sialic acid in a single reaction. In preferred embodiments, commercial scale refers to production of greater than about 50, 75, 80, 90 or 100, 125, 150, 175, or 200 grams. An expression cassette can be constructed for production of more than one protein. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette. For cells, saccharides, nucleic acids, and polypeptides of the invention, the term “isolated” refers to material that is substantially or essentially free from components which normally accompany the material as found in its native state. Plasmids containing one or more of the above listed components employs standard ligation techniques as described in the references cited above. At last, such genetically engineered micro-organisms are not only viable but they are able to grow in standard conditions. Other transformation methods are also suitable.<br>
<br> A “culture medium” refers to any liquid, semi-solid or solid media that can be used to support the growth of a microorganism used in the methods of the invention. Media can be rich media, e.g., Luria broth or terrific broth, or synthetic or semi-synthetic medium, e.g., M9 medium. Alternatively, selectable markers may encode proteins that complement auxotrophic deficiencies or supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Those of skill are aware that insertion of a nucleic acid into a chromosome can occur, e.g., by homologous recombination. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure as measured by band intensity on a silver stained gel or other method for determining purity.<br>
<br> Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous sequence. The nanT, nanA, nanK and nanE genes are part of the same operon, which is regulated by the DNA binding protein NanR and induced by Neu5Ac (Kalivoda et al., 2003). The production of Neu5Ac by the NeuB and NeuC proteins can thus induce the pathway of Neu5Ac catabolism and create two futile cycles that reduce the capacity of CMP-Neu5Ac biosynthesis of the bacteria. In a first embodiment, the invention relates to a method for producing sialic acid and analogs thereof, comprising the step consisting of culturing a microorganism in a culture medium, wherein said microorganism comprises heterologous genes encoding a sialic acid synthase (NeuB), a UDP-GlcNAc epimerase (NeuC), said micro-organism being devoid of a gene encoding CMP-Neu5Ac synthase (NeuA) or wherein a gene encoding CMP-Neu5Ac synthase (NeuA) has been inactivated or deleted; and wherein endogenous genes coding for sialic acid aldolase (NanA), for sialic acid transporter (nanT), and optionally for ManNac kinase (nanK), have been deleted or inactivated.<br>
<br> Alternatively, the method may comprise removing the operon including nanT, nanA, nanE genes (nanEAT-), except the nanK gene. Thus, the expression of neuB and neuC genes in a bacteria results in an accumulation of Neu5Ac if the bacteria is devoid of CMP-Neu5Ac synthase activity. A “recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of affecting expression of a structural gene in hosts compatible with such sequences. 1977) Nucleic Acids Res. 50 residus in length, more preferably over a region of at least about 100 residus, and most preferably the sequences are substantially identical over at least about 150 residus. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residus in length, more preferably over a region of at least about 100 residus, and most preferably the sequences are substantially identical over at least about 150 residus. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. “silent substitutions” or “silent variations,” which are one species of “conservatively modified variations.” Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.<br>

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