Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-01-07
2004-12-28
Whisenant, Ethan (Department: 1643)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S024100, C435S006120, C435S191000, C435S320100
Reexamination Certificate
active
06835820
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to nucleic acid and amino acid sequences involved in polyhydroxyalkanoate biosynthesis, and more specifically, to polyhydroxyalkanoate biosynthesis sequences isolated from
Bacillus megaterium
. In particular, nucleic acid sequences phaP, phaQ, phaR, phaB, phaC, and their encoded amino acid sequences are disclosed.
BACKGROUND OF THE INVENTION
Polyhydroxyalkanoic acids (PHA) are a class of aliphatic polyesters that accumulate in inclusion-bodies in many bacteria and archaea (2, 41). Their physiological role in the cell is that of carbon and energy reserves, and as a sink for reducing power. The most studied PHA have repeating subunits of: —[O—CH(R)(CH
2
)
x
CO]—, where the most common form is polyhydroxybutyrate (PHB), with R=CH
3
and x=1 (45). The PHA biosynthetic pathway has been determined for
Alcaligenes eutrophus
(17, 18, 44). In this organism two molecules of acetyl-Coenzyme A (CoA) are condensed by &bgr;-ketothiolase (PhaA), followed by a stereo-specific reduction catalyzed by an NADPH dependent acetoacetyl-CoA reductase (PhaB) to produce the monomer D-(−)-&bgr;-hydroxybutyryl-CoA, which is polymerized by PHA synthase (PhaC). These 3 pha genes are coded on the phaCAB operon, which is speculated to be constitutively expressed, but PHA is not constitutively synthesized. Alternative pathways for synthesis of the monomer in other organisms have been suggested, most notably in the Pseudomonas species where the side chain, R, is longer than CH
3
and its composition is influenced by carbon substrates in the growth medium (7, 45). In addition to
A. eutrophus
, phaC has been cloned from more than twenty different bacteria (26, 43). Other genes associated with PHA synthesis, phaA, phaB, phaZ (PHA depolymerase) and genes for inclusion-body associated proteins and other low molecular weight proteins of unknown function, have also been cloned from some of these bacteria, in many cases by virtue of the fact that they are clustered with phaC.
PHA inclusion-bodies are 0.2 to 0.5&mgr;m in diameter, but their structural details are largely unknown. They were described originally for some species of Bacillus (6, 8, 15, 30, 47) and later for many more bacteria including Pseudomonas, Alcaligenes and Rhodococcus (5, 11, 12, 25, 42). Those from
Bacillus megaterium
were shown to contain 97.7% PHA, 1.87% protein and 0.46% lipid with protein and lipid forming an outer layer (15). More recent reports show the presence of a 14 kDa protein (GA14) on PHA inclusion-bodies of
R. ruber
(36, 37), and a 24 kDa protein (GA24) with similarities to GA14 on the inclusion-bodies of
A. eutrophus
(48). These proteins are not essential for PHA accumulation but have been shown to influence the size of PHA inclusion-bodies and the rate of PHA accumulation (37, 48). GA14 and GA24 have been named “phasins” due to some similarities with oleosins, which are proteins on the surface of oil bodies in plant seeds (21). Granule associated proteins are wide-spread in PHA accumulating bacteria (49).
The pattern of PHA inclusion-body growth and proliferation throughout the growth cycle of
Bacillus megaterium
has been described (32).
There exists a need for additional nucleic acid and amino acid sequences useful for the production of polymers in biological systems.
SUMMARY OF THE INVENTION
This invention is the result of a study of PHA inclusion-body associated proteins from
Bacillus megaterium
and the cloning and analysis of their coding region. The transcription starts were identified, the functional expression of several of the sequences was confirmed in
Escherichia coli
and in PHA negative mutants of
Bacillus megaterium
and
Pseudomonas putida
, and PhaP and PhaC were localized to PHA inclusion-bodies throughout growth.
A nucleic acid fragment encoding proteins involved in polyhydroxyalkanoate biosynthesis was isolated from
Bacillus megaterium
. Nine nucleic acid sequences and their encoded amino acid sequences are disclosed. Sequences encoding PhaB and PhaC display not insignificant percent identity and similarity to known acetoacetyl-CoA reductase and polyhydroxyalkanoate synthase proteins, while sequences encoding PhaP, PhaQ, and PhaR do not display significant similarity to known sequences. YkoY is similar to known toxic anion resistance proteins; YkoZ is similar to known RNA polymerase sigma factors; YkrM is similar to known Na
+
-transporting ATP synthase proteins; and SspD matches the known
B. megaterium
spore specific DNA binding protein.
While several PHA related sequences were expressed in two organisms, it is envisioned that the sequences may be expressed in a wide array of organisms, and that the nucleic acid sequences themselves may be modified to change the sequence and properties of the encoded proteins.
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Cannon Francis C.
Cannon Maura
Gruys Kenneth J.
McCool Gabriel J.
Valentin Henry E.
Chakrabarti Arun Kr.
Holland & Knight LLP
The University of Massachusetts
Whisenant Ethan
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