Recombinant cellulose synthase

Details Recombinant cellulose synthase Recombinant cellulose synthase

1995-10-10

2003-04-01

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S193000, C435S194000, C435S252330, C435S257200, C435S242000, C435S242000, C435S242000, C435S320100

Reexamination Certificate

active

06541238

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to compositions of and methods for obtaining cellulose synthase. The invention relates as well to the DNA sequences encoding cellulose synthase, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors and recombinant cellulose synthase polypeptides. More specifically, the invention relates to the cloning and expression of cellulose synthase from
Acetobacter xylinum.
2. Description of the Related Art
Cellulose biosynthesis is an event largely associated with plant cells, especially certain agronomic species such as cotton where as much as 90-95% of the secondary wall of mature cotton fibers is composed of cellulose. However, studies of cellulose biosynthesis in higher plants have been very frustrating, mainly because cellulose synthase, due to its lability, has evaded isolation and purification. Furthermore, the study of this enzyme has been hampered by the great difficulty in detecting formation of cellulose in vitro using purified preparations of plant and other eukaryotic cells [Delmer 1987: In higher plant preparations, the &bgr;-1,3-glucan (callose) chains are synthesized instead of the &bgr;-1,4-glucan of cellulose].
Apart from the higher plants, a large number of bacteria synthesize cellulose, including
Acetobacter xylinum
(
A. xylinum
), which converts as much as 35% of the glucose supplied to cultures of this bacterium into cellulose. Unlike the situation in plants and other eukaryotic cells noted above, the cellulose synthase of this bacterium has been isolated to a certain degree. Because of these facts, coupled with the capacity of the bacterium to synthesize a highly purified form of cellulose in vitro using membrane preparations,
A. xylinum
has become a preferred system for studies on the synthesis and organization of cellulose [Delmer 1987: In the bacterium, &bgr;-1,4-glucan (cellulose) chains can be detected].
The biosynthesis of cellulose in
A. xylinum
is visualized as a two-step process. The first step involves the polymerization of sugar nucleotides (UDP-glucose; UDP-glc) into a &bgr;-1,4,-linked glucan chain. The polymerization reaction is catalyzed by the enzyme cellulose synthase (UDP-glucose:1,4-&bgr;-D-glucosyltransferase; E.C. 2.4.1.12) which is present in the cytoplasmic membrane (Bureau and Brown 1987). The activity of this enzyme is regulated by bis-(3′, 5′)-cyclic diguanylic acid (Ross 1987).
Purification of the cellulose synthase activity from membrane preparations has been accomplished by an entrapment procedure (Lin and Brown 1989) similar in some aspects to one used in the purification of chitin synthase from yeast (Kang 1984). The molecular weight of the native enzyme in Triton X-100 solubilized preparations appears to be 490 kd as determined by gel filtration (Lin and Brown 1989). Electron microscopy of the purified preparation shows doughnut-shaped particles indicating that the cellulose synthase may be organized as a tetramer or octamer (Lin and Brown 1989). Lithium dodecyl sulfate polyacrylamide gel electrophoresis of the purified preparation shows two major bands with molecular weights of 93 kd and 83 kd. The resistance of the cellulose synthase activity and of the 83 kd polypeptide to trypsin treatment has suggested that the 83 kd polypeptide is the active cellulose synthase (Lin and Brown 1989).
Photoaffinity probes have been used with glucan synthases to identify these enzymes in a variety of different species. In red beets, a 57 kd polypeptide has been shown to be the substrate-binding component of (1,3)-&bgr;-glucan synthase (Wasserman 1989). In cotton fibers and in mung bean, a 50 kd polypeptide appears to bind the substrate (Delmer and Solomon 1989). In
A. xylinum
, Mayer et al. (1989) suggested a 67 k polypeptide as the substrate-binding subunit and a 57 kd polypeptide as the activator-binding subunit of a 420 kd oligomeric cellulose synthase. These investigators suggested the presence of similarly sized peptides in other cellulose-producing organisms such as
Agrobacterium tumefaciens
, mung bean, wheat, pea and cotton on the basis of immunochemical analyses.
Genes involved in the synthesis of other exopolysaccharides in a number of bacteria have been cloned using the standard approach of genetic complementation [Easson (1987); Harding (1987); Calvin and Hanawalt (1988)]. Mutants of
A. xylinum
defective in the production of cellulose, yet which still possess a normal complement of cellulose synthase have been identified in particular strains. Certain of these mutants have been shown to be deficient in the activity of UDPG-pyrophosphorylase, the enzyme required for the synthesis of the cellulose synthase substrate molecule, UDP-glucose. Complementation of these mutants by cloned fragments from
A. xylinum
-derived DNA has led to the isolation of the gene encoding UDPG-pyrophosphorylase (Valla 1989).
Surprisingly, however, no mutants actually deficient in cellulose synthase activity have been identified. This is despite the fact that there have been a large number of mutants identified which morphologically appear to be cellulose-deficient, but which synthesize small amounts of an altered crystalline polymorph of cellulose (cellulose II) and have the wild type level of cellulose synthase activity when assayed in vitro for cellulose synthesis [Saxena and Brown (1989); Roberts et al. (1989)].
Thus, it has been considerably difficult to apply the standard approaches of classical genetics or even the powerful techniques of molecular biology to the study of cellulose biosynthesis. Lacking purified quantities of the enzyme, lacking detailed information of the subunit architecture, lacking means to specifically identify the substrate-binding subunit, and lacking a simple genetic method of isolating mutants deficient in cellulose synthase activity, have each hampered the ability of researchers to isolate DNA segments encoding cellulose synthase.
SUMMARY OF THE INVENTION
The present invention for the first time, provides the vectors, DNA segments, purified protein, antibodies, methods of cloning, and recombinant host cells, seeds and plants necessary to obtain and use a recombinant cellulose synthase. Thus, the difficulties encountered with applying the standard approaches of classical genetics or techniques of molecular biology to the study of cellulose biosynthesis have been overcome. Accordingly, the present invention concerns generally compositions and methods for the preparation of recombinant cellulose synthase of both prokaryotic and eukaryotic origin.
In certain general and overall embodiments, the invention concerns recombinant vectors and isolated DNA segments encoding a cellulose synthase peptide. The DNA segments of the invention may encode biologically functional equivalent protein or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of the amino acids being exchanged.
In the context of the present invention, the term cellulose synthase is intended to refer to peptides or proteins having the biological and the immunological identity of the cellulose synthase of the cell enabled lines by the present invention. For example, such cell lines would include cells of
Acetobacter xylinum
. Generally, the cellulose synthase of the invention will refer to a 723 amino acid peptide or protein (SEQ ID NO:2) in that this is the precise length of the only presently sequenced cellulose synthase. However, the invention does not preclude and, in fact enables, preparation or use of shorter or longer peptides or proteins, so long as a peptide or protein has similar in kind biological activity and/or a cross reactive immunological reactivity, for example, as defined by rabbit polyclonal antisera. For instance, the other wild type strain of
A. xylinum
ATCC 23769 which was used by the present inventors possesses a 75 kD polypeptide as a catalytic

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