The invention relates to a microbial cell, typically a bacterial cell, genetically engineered to produce a modified sugar nucleotide, for example UDP glucuronic acid, and the use of the modified sugar to transfer glucuronic acid to small acceptor molecules.
Glycosyltransferases (GTases) are enzymes that post-translationally transfer glycosyl residues from an activated nucleotide sugar to monomeric and polymeric acceptor molecules such as other sugars, proteins, lipids and other organic substrates. These glycosylated molecules take part in diverse metabolic pathways and processes. The transfer of a glycosyl moiety can alter the acceptor's bioactivity, solubility and transport properties within the cell and throughout the organism. The most common activated nucleotide sugar is UDP-glucose which is used by a large number of glucosyltransferase enzymes. Examples of other GTases include rhamnosyltransferases, fucosyltransferases, sialyltransferases, galatosyltransferases and glucuronosyltransferases, each of which use a different donating nucleotide sugar.
Glucuronosyltransferases catalyse the transfer of glucuronic acid from UDP glucuronic acid to small molecule acceptors in both mammalian and plant systems. Typically the small molecules are drugs, environmental chemicals and endogenous substances. In humans the glucuronosyltransferase are grouped into two families; UGT1 and UGT2. UGT1 family members are typically involved in the modification of phenol substrates and bilirubin and some members can modify oestrogens. UGT2 enzymes are subdivided into UGT2A and UGT2B. UGT2A includes genes that encode glucuronosyltransferases expressed by the olfactory epithelium and UGT2B includes genes that encode glucuronosyltransferases that modify bile acids, C19 steroids, C18 steroids, fatty acids, carboxylic acids, phenols and carcinogens. Several members of the UGT2B sub-family have been isolated, for example U.S. Pat. No. 6,287,834 discloses a human glucuronosyltransferase referred to as the UGT2B17. A further example of the isolation and characterisation of glucuronosyltransferase genes is disclosed in WO2006028985.
Plant glucuronosyltransferases are also known. For example, Woo et al (Plant Cell, vol 11, 2303-2315, 1999) describe a Pisum sativum UDP glucuronosyltransferase that is thought to modify flavonoids. The essential function of this glucuronosyltransferase is illustrated by antisense experiments in Medicargo sativa and Arabidopsis thalina which showed cell cycle effects resulting in delayed root emergence, reduced root growth and increased lateral root development (Woo et al Plant Physiology vol 133, 538-548, 2003).
Bacterial expression systems for the production of small molecules, in particular antioxidants, amino acids and peptides, are well known in the art. Typically, bacterial host cells are transformed with a vector that is provided with expression signals that are operably linked to a nucleic acid molecule that encodes a polypeptide sequence the expression of which is desired. Vectors are also provided with replication origins that facilitate the replication of the vector inside the host bacterium. UDP-glucuronic acid is the main nucleotide sugar used by glucuronosyltransferases in mammalian cells for conjugating and detoxifying xenobiotics and steroids. In plants, this sugar nucleotide acts as a precursor for cell wall synthesis. UDP-glucuronic acid is synthesized from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UGD). We disclose the expression of UGD genes in an E. coli system to increase the level of UDP-glucuronic acid in the transformed bacterial cells. This system can be used for production of UDP-glucuronic acid on its own as well as for production of glucuronides when applied in conjunction with glucuronosyltransferases capable of recognising UDP-glucuronic acid as an activated sugar-donor.
According to an aspect of the invention there is provided a microbial cell wherein said cell is genetically modified which modification is the transformation of said cell with a nucleic acid molecule wherein said nucleic acid molecule encodes a polypeptide with the specific enzyme activity associated with a UDP-glucose dehydrogenase.
In a preferred embodiment of the invention said enzyme activity is over-expressed when compared to a non-transformed reference cell of the same species.
In a preferred embodiment of the invention said nucleic acid molecule is selected from the group consisting of:
i) a nucleic acid molecule comprising a nucleic acid sequence as represented by FIG. 1a, 1b, 1c or 1d;
ii) a nucleic acid molecule which hybridises to the nucleic acid molecule in (i) and which has the enzyme activity associated with UDP-glucose dehydrogenase.
In a further preferred embodiment of the invention said nucleic acid molecule hybridises under stringent hybridisation conditions to the sequence presented in FIG. 1a, 1b, 1c or 1d.
Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, N.Y., 1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (allows sequences that share
at least 90% identity to hybridize)
5x SSC at 65° C. for 16 hours
2x SSC at room temperature (RT) for 15 minutes each
0.5x SSC at 65° C. for 20 minutes each
High Stringency (allows sequences that share
at least 80% identity to hybridize)