Processive sugar transferase

Details Processive sugar transferase Processive sugar transferase

2000-09-22

2004-06-01

Rao, Manjunath (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S004000, C435S041000, C435S069100, C435S183000, C435S193000, C435S252300, C435S254110, C435S254200, C435S410000, C435S320100, C435S419000, C536S023200, C536S023700

Reexamination Certificate

active

06743606

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the use of processive UDP-sugar: 1,2-diacylglycerol-3-&bgr;-, UDP-sugar: 3-&bgr;-1,3′-phospho-sn-glycerol-1′,2′-diacyl-sn-glycerol)- and UDP-sugar: 3-[O-&bgr;-D-glucopyrartosyl]-sn-glycerol-1,3 ′-phospho-1′,2′-diacyl-sn-glycerol-D-sugar transferases and similar proteins as well as the corresponding coding nucleic acids for the manipulation of the contents and/or the structure of glycosyldiacylglycerols and/or the synthetic secondary products thereof, as well as other substrates which are glycosylated by these enzymes, in transgenic cells and/or organisms.
Glycosyldiacylglycerols were produced enzymatically by means of a sugar transferase (glycosyl transferase). For this purpose, the gene coding for a UDP-sugar transferase was isolated from genomic DNA of
Bacillus subtilis
and
Staphylococcus aureus
, and cloned into, and expressed in,
E. coli
. The activity of the enzymes was confirmed by means of specific in vitro-enzyme assays. The products were also detected and identified in lipid extracts of transgenic
E. coli
cells. The products are various novel glycolipids having different number of glucose residues (maximum of 4) linked via a &bgr;(1→6)glycosidic bond, and utilizing diacylgylcerol (DAG) or phosphatidylglycerol (PG) as the primary acceptor.
In addition, these novel glycolipids comprise two differently structured novel phosphoglycolipids (PL1 and PL2) with a different number of glucose residues (maximum of two) which in the case of (PL2) are also linked via a &bgr;(1→6)glycosidic bond and utilize phosphatidylglycerol as the acceptor (i.e. both diastereomers, i.e. with respect to the configuration of the non-acylated glycerol residue). The glycosyl residues may further be acylated in position 6′″ of the terminal glucose:
1) MGlcD: 3-[O-&bgr;-D-glucopyranosyl]-1,2-diacylglycerol (
Staphylococcus aureus
ypfP)
2) DGlcD: 3-[O-&bgr;-D-glucopyranosyl-(1→6)-O-&bgr;-D-glucopyranosyl]-1,2-diacylglycerol
3) TGlcD: 3-[O-&bgr;-D-glucopyranosyl-(1→6)-O-&bgr;-D-glucopyranosyl-(1→6)-O-&bgr;-D-glucopyranosyl]-1,2-diacylglycerol
4) TeGlcD: 3-[O-&bgr;-D-glucopyranosyl-(1→6)-O-&bgr;-D-glucopyranosyl-(1→6)-O-&bgr;-D-glucopyranosyl-(1→6)-O-&bgr;-D-glucopyranosyl]-1,2-diacylglycerol
5) Phospholipid 1:3-[O-&bgr;-D-glucopyranosyl]-sn-glycerol-1,3′-phospho-1′,2′-diacyl-sn-glycerol)
6) Phospholipid 2:{3-[O-(6′″-O-acyl)-&bgr;-D-glucopyranosyl-(1′″6′)-O-&bgr;-D-glucopyranosyl]-2-acyl-sn-glycerol-1,3′-phospho-1′,2′-diacyl-sn-glycerol}
Note: The numbering of the glycerol residues I (Gro
I
) and II (Gro
II
) corresponds herein to the numbering 1-3 and 1′-3′, respectively, i.e. Gro
I
is “left-hand” and Gro
II
is “right-hand” in accordance with FIG.
13
.
Surprisingly, the enzymes act in a processive manner, i.e. all detected novel glycolipids are formed by successive addition of UDP-glucose to the respective preceding product of the enzymes. Further, alkyl-&bgr;-D-glucosides, ceramides (both enzymes), sterols and sterol glucosides (only the enzyme of
S. aureus
) are used as acceptors for a further glucosylation reaction.
DETAILED DESCRIPTION
Glyceroglycolipids represent a group of membrane components which are very hetero-geneous with respect to their structure. They are found in bacteria (Kates, 1990), plants and in very low amounts also in animals. Many structures especially of bacterial glycolipids have already been described many years ago (Kates, 1999), however, none of the genes synthesizing these glycolipids have been cloned, so that these substances can be obtained from the corresponding organisms only in analytical amounts. Only at the beginning of 1997 was the first publication issued, wherein the cloning and expression of a plant galactose: 1,2-diacylglycerol galactosyl transferase is described (Shimojina et al., 1997). However, this enzyme is no “processive” glycosyl transferase.
Database searches in the “U.S. Patent Database” revealed that two further patents relating to glycosyl transferases exist: U.S. Pat. No. 5,545,554: Glycosyl transferases for biosynthesis of oligosaccharides, and genes and encoding them, and U.S. Pat. No. 5,641,668: Proteins having glycosyl transferase activity. It appears that the first-mentioned patent only relates to glycosyl transferases which synthesize oligosaccharide, so that this patent is not relevant with respect to the enzyme, viz a lipid glycosyl transferase, described herein. The second-mentioned patent relates to glycosyl transferases in general, in view of which the processive enzyme described in this specification is novel.
BRIEF DESCRIPTION OF THE FIGURES
Glycosyl diacylglycerols are naturally occurring compounds found in plants, animals and bacteria. However, an inexpensive, large-scale production of these compounds was not possible so far, since corresponding genes were not yet cloned. Glycosyl diacylglycerols can be used in a variety of applications, depending on the number of sugar residues and the structure of the fatty acids.
When esterified with usual C18 unsaturated fatty acids, diglucosyl diacylglycerols have emulsifier properties which are useful in food industrial applications (in mayonnaise, margarine, ice cream, confectionery etc.).
In the presence of highly unsaturated fatty acids, glycolipids may be introduced into polymers, which then obtain new characteristics and surfaces. Finally, glycosyl diacylgylcerols may obtain detergent characteristics, when the fatty acid chain length is drastically shortened. This would already now be possible in transgenic rape seed with predominant lauric acid. Such detergents could be produced in large amounts in an inexpensive manner, and such detergents would be biologically degradable.
The phospholipids which are glucosylated by the enzyme of
S. aureus
receive new physico-chemical characteristics due to the charge of the phosphate residue between the two glycerol residues on the one hand, and on the other hand due to acylation of the sugar residue(s). Thus, by use of the described processive sugar transferases, not only neutral lipids, but also charged glycolipids can be specifically produced and varied. Thus, a further class of charged glycophospholipids are developed via the sugar transferases.
In the production of plant oils from oil seeds, a lecithin fraction is obtained, wherein phospholipids and glycolipids are accumulated. By over-expressing the genes disclosed in this specification in these plants, a variety of glycolipids (glucosyl diacylglycerols, steryl glucoside, glucocerebroside and other lipids described herein) could be concentrated, with a favourable effect on the baking properties of bakery products, to which the lecithin fraction is added.
In addition, the phospholipids glucosylated by the
S. aureus
enzyme receive further physicochemical properties due to the charge of the phosphate residue.
This invention, therefore, relates to a process for the production of glycolipids in transgenic cells and/or organisms, comprising the following steps:
transfer of a nucleic acid molecule that codes for a protein having the biological activity of a processive diacylglycerol glycosyltransferase to the cells or organism,
expression of the protein having a biological activity of a processive diacylglycerol glycosyltransferase under suitable regulatory sequences in the cells or the organism, and
if desired, recovery of the glycolipids synthesized by the biological activity of a processive diacylglycerol glycosyltransferase from the cells or the organism.
In a preferred embodiment of the invention, the nucleic acid molecule codes for a protein having the biological activity of a processive diacylglycerol glycosyltransferase from
Bacillus subtilis
or
Staphylococcus aureus.
The transgenic cells may be any cells that are useful for the production of the new glycolipids, preferably the cells are pl

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