Bacterial sucrose synthase compositions and methods of use

Details Bacterial sucrose synthase compositions and methods of use Bacterial sucrose synthase compositions and methods of use

1996-07-19

2004-01-27

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

Reexamination Certificate

active

06682918

ABSTRACT:

1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates to the field of molecular biology. More specifically, it concerns nucleic acid compositions comprising bacterial sucrose synthases, methods for making and using native and recombinant sucrose synthase-encoding polypeptides, and methods for making and using polynucleotides encoding sucrose synthase polypeptides.
1.2 Description of the Related Art
1.2.1 Sucrose Synthase
Sucrose synthase (EC 2.1.4.13) is an enzyme that is found in plants and cyanobacteria. It catalyzes the reversible reaction:
UDP-glucose+fructose⇄sucrose+UDP
In plants, sucrose synthase is mostly active in sink tissues such as tubers, seeds, fruits and meristems where it catalyzes the breakdown of phloem-transported sucrose from the leaves to UDP-glucose and fructose. Subsequent reactions in storage tissues utilize the UDP-glucose directly to generate starch using the enzyme UDP-glucose pyrophosphorylase. In other sink tissues, the monosaccharides may accumulate (fruits) or be utilized for energy or growth (roots, meristems). Multiple sucrose synthase alleles have been identified in several plants, and typically display tissue-specific expression (Huang et al., 1996; Choury et al., 1986). Expression levels of sucrose synthase in sink tissues is thought to be the main indicator of sink strength (Amor et al., 1995; Zrenner et al., 1995).
Regulation of sucrose synthase appears to be active at several levels, including transcriptional control, feedback inhibition by glucose and fructose, and transcriptional mechanisms (Geigenberger and Stitt, 1993). Regarding the latter, it has recently been determined that sucrose synthase in maize is reversibly phosphorylated at a serine residue near the N-terminus of the protein, and this site is strongly conserved among all the plant sequences. Similarly sucrose phosphate synthase has also been shown to be controlled by phosphorylation at several serine residues (Huber and Huber, 1996).
1.2.2 Sucrose Phosphate Synthase
Formation of sucrose in source tissues, such as mature leaves, utilizes a different enzyme, sucrose-phosphate synthase (EC 2.4.1.14), which catalyzes the reaction:
UDP-glucose+fructose 6-phosphate⇄sucrose 6-phosphate+UDP
A subsequent step catalyzed by sucrose phosphatase (EC 3.1.3.24) removes the phosphate from sucrose 6phosphate, essentially making this reaction irreversible. The sucrose is then transported into the phloem of the plant utilizing a sucrose-proton symporter.
1.2.3 Bacterial Sucrose Synthase Differs From the Plant Enzyme
In the filamentous cyanobacterium Anabaena sp. strain PCC 7120 there is evidence that sucrose is synthesized in vegetative cells and is transported to special differentiated cells called heterocysts, where it supports nitrogen fixation. This evidence is based on observations of a sucrose synthase activity in crude extracts that co-purifies with vegetative cells but not heterocysts (Schilling and Ehrnsperger, 1985). An alkaline invertase activity (for the degradation of sucrose) has been identified which copurifies with heterocysts but not vegetative cells. This work suggests that sucrose synthase is responsible for the synthesis of sucrose that is then transported into the heterocyst where it is degraded to glucose and fructose by alkaline invertase. While the role of sucrose synthase in most plant tissues is the breakdown of sucrose, the enzyme has been demonstrated to be freely reversible (Geigenberger and Stitt, 1993), and may function in the synthesis of sucrose in cyanobacteria.
1.2.4 Deficiencies in the Prior Art
The genetic transformation of important commercial agricultural crops with DNA segments encoding sucrose synthase enzymes would be a revolution in the farming of such grains as wheat, rice, maize, barley, rye, and oats. Moreover the availability for modulating the starch and/or sucrose content in plants such as potatoes, tomatoes, fruits such as apples, cherries, pears, strawberries and raspberries would be highly desirable. The ability to modulate nitrogen fixation activity in plants such as soybean, alfalfa, beans, peas, and related legumes would also represent a breakthrough in the areas of improving crop yields where fixed-nitrogen fertilizer input is limited.
Therefore, what is needed in the art are compositions comprising bacterial sucrose synthase-encoding DNA segments and sucrose synthase polypeptides, as well as methods for the alteration of sucrose synthase activity in vitro and in vivo. Methods of identifying and assaying the levels of sucrose synthase activity in plants, fungi, bacteria and cyanobacteria would also be important in genetically engineering cells for altered sucrose and starch production and nitrogen fixation activity.
Moreover, what is lacking in the prior art is the identification of DNA segments encoding bacterial and, particularly, cyanobacterial sucrose synthase enzymes, and the development of methods and processes for their use in creation of modified, transgenic plants which have altered sucrose synthase activity. Moreover, novel methods providing transgenic plants using DNA segments encoding sucrose synthase polypeptides to modulate starch and sucrose biosynthesis in general, and nitrogen fixation activity of cells in specific, are greatly needed to provide transformed plants altered in such activities. Methods for determining sucrose synthase activity in vivo and quantitating the level of sucrose synthase expression in bacteria and transformed plants would also represent major improvements over the current state of the art.
2. SUMMARY OF THE INVENTION
The present invention seeks to overcome these and other inherent deficiencies in the prior art by providing compositions comprising novel sucrose synthase polypeptides from bacterial, and particularly, cyanobacterial species. The invention also provides novel DNA segments encoding prokaryotic sucrose synthases, and methods and processes for their use in regulating the starch and/or sucrose content of plant tissues, for conferring and modulating nitrogen fixation activity in a variety of different cell types, and for altering the activity of sucrose synthase in plant cells in vivo. Also disclosed are methods for determining sucrose synthase activity and expression, and kits for identifying the presence of sucrose synthase polypeptides and DNA segments which encode them.
The bacterial sequences of the present invention differ markedly from eukaryotic enzymes which catalyze the same reaction in higher organisms such as plants. The bacterial proteins disclosed herein have less than 44% sequence homology on average to the eukaryotic proteins, and the nucleic acid sequences encoding the bacterial enzymes are less than 56% identical to plant cDNAs encoding eukaryotic sucrose synthase proteins. The longest contiguous nucleic acid sequence which is identical to any of the sequences in the prior art encoding sucrose synthases is less than 14 residues, suggesting broad differences exist between the novel sequences disclosed herein, and the eukaryotic sequences disclosed in the prior art.
Dramatic differences between prokaryotic and eukaryotic sucrose synthases have been identified by the present inventors in the protein sequences, particularly in the amino terminal region of the proteins. In plants, it has been demonstrated that significant protein homologies exist between plant proteins presumably since the region is a site for protein phosphorylation in eukaryotic species. In sharp contrast, no such phosphorylation site is observed in the prokaryotic sequences disclosed herein, and little amino acid identity is observed. In fact, in the first 20 amino acid residues, virtually no similarity exists to any plant-derived protein.
2.1 Sucrose Synthase Genes and Polynucleotides
The present invention provides polynucleotides and polypeptides relating to a whole or a portion of sucrose synthase of a bacterium, and particularly, the sucrose synthase of a cyanobacterium, as well as processes for making, using, detecting and modulating th

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