Polyhydroxyalkanoate synthase genes

Details Polyhydroxyalkanoate synthase genes Polyhydroxyalkanoate synthase genes

2000-09-28

2002-11-05

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C800S288000, C800S281000, C800S280000, C435S005000, C435S091100, C435S091200, C435S173300, C435S135000, C435S069100, C435S320100, C536S023700

Reexamination Certificate

active

06475734

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to genes encoding enzymes involved the biosynthesis of biodegradable thermoplastics known as polyhydroxyalkanoates.
BACKGROUND OF THE INVENTION
The production of intracellular polyesters belonging to the class of polymers known as polyhydroxyalkanoates (PHAs) has been observed in a wide array of prokaryotic organisms. PHAs are bacterial polyesters that accumulate in a wide variety of bacteria. The polymers are biodegradable and are an attractive source of nonpolluting plastics and elastomers. The monomers of the polyesters range in length from C
4
to C
12
. PHAs are broadly characterized according to the monomers that constitute their backbone.
PHA synthase genes have been characterized from about 30 bacteria. The genes can be divided into two classes based upon the substrate specificity towards 3-hydroxyalkanoate-CoA. Class I accepts short-chain-length (SCL) 3-hydroxyalkanoate-CoA from about C
4
to about C
6
and class II accepts medium-chain-length (MSL) 3-hydroxyalkanoate-CoA from about C
6
to about C
4
. Only a few exceptions exist. For example, a PHA synthase from
Thiocapsa pfennigii
can produce PHA from C
4
to C
8
(Liebergesell et al. 1993; WO 96/08566) and a PHA synthase from Pseudomonas sp. 61-3 can synthesize PHA from C
4
to C
12
(Matsusaki et al. (1998)
J. Bacteriol
. 180:6459-6467).
Lee et al. ((1995)
Appl. Microbiol. Biotechnol
. 42:901) compared PHA production of several Pseudomonas strains.
Pseudomonas fluorescens
strain GK13 and Pseudomonas sp. A33 showed an unusual poly(3HBcoX) composition pattern. With 1,3-butanediol as a carbon source, this strain produced PHA with a composition of 15.1 mol % 3HB (3-hydroxybutyric acid), 3.5 mol % 3HHx (3-hydroxyhexanoate), 15.7 mol % 3HO (3-hydroxyoctanoate) and 65.7 mol % 3HD(3-hydroxydecanoate).
P. fluorescens
strain GK13 and Pseudomonas sp. A33 showed identical hybridization patterns when restricted DNA was hybridized employing labeled oligonucleotide probe highly specific for PHA synthases. A 12.5-kbp genomic EcoRI fragment from Pseudomonas sp. A33 conferred the ability for poly(3HBcoX) synthesis to a PHA negative mutant of
Ralstonia eutropha
. With gluconate as a carbon source, the transformed Ralstonia strain produced PHA with a composition of 89.9 mol % 3HB and 10.1 mol % 3HD. Based upon the similarities of strain A33 and GK13 concerning PHA synthesis and hybridization pattern, a 12.5-kbp genomic EcoRI fragment from strain GK13 most probably encodes for a PHA synthase, which is able to synthesize poly(3HBcoX). The only other example of a poly(3HBcoX)-synthesizing PHA synthase was reported by Matsusaki et al. ((1998)
J. Bacteriol
. 180:6459-6467).
The polymerization of the hydroxyacyl-CoA substrates is carried out by PHA synthases. The substrate specificity of this class of enzymes varies across the spectrum of PHA producing organisms. The variation in substrate specificity of PHA synthases is supported by indirect evidence observed in heterologous expression studies (Lee et al. (1995)
Appl. Microbiol. Biotechnol
. 42:901 and Timm et al. (1990)
Appl. Microbiol. Biotech
. 33:296). Hence, the structure of the backbone of the polymer is strongly influenced by the PHA synthase responsible for its formation.
SUMMARY OF THE INVENTION
Compositions and methods for the production of PHA in plants and host cells are provided. Particularly, isolated nucleotide molecules comprising nucleotide sequences encoding PHA synthases with broad substrate specificity are disclosed from
Pseudomonas fluorescens
strain GK13 (DSM7139). Additionally provided are isolated polypeptides comprising the amino acid sequences of such PHA synthases. The nucleotide molecules of the invention can be used to produce, in plants and other organisms, poly(3HBcoX), where X has an acyl chain length of greater than or equal to C
8
. The PHA synthases of the invention can be targeted to the peroxisomes in plants by operably linking peroxisomal targeting sequences to the nucleotide sequences encoding the PHA synthases. In this manner, the invention provides for the production of PHA copolymers in plant peroxisomes. The nucleotide sequences of the invention can be used in combination with other sequences for the production of novel biodegradable polyesters in plants.
Transformed host cells, plants, plant tissues, plant cells and seeds are provided.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for the production of biodegradable polyesters in plants and other organisms are provided. In particular, isolated nucleotide molecules comprising nucleotide sequences for PHA synthase genes, particularly, phaC1 and phaC2 from
Pseudomonas fluorescens
GK13, are provided (SEQ ID NOs: 1 and 3, respectively). The sequences find use in plants and other organisms for the production of PHA, particularly PHA copolymers, more particularly poly(3HBcoX). By “poly(3HBcoX)” is intended a PHA copolymer comprised of 3-hydroxybutyrate (3HB) and any other hydroxyalkanoate, designated herein as X.
The nucleotide sequences of the invention can be used in combination with other sequences including, but not limited to nucleotide sequences encoding &bgr;-ketothiolase, acetoacetyl-CoA reductase, the R-specific enoyl-CoA hydratase domain of the yeast multifunctional protein (MFP), enoyl-CoA hydratase, and 3-hydroxyacyl-ACP CoA-transferase (phaG). The sequences can be provided with peroxisome-targeting sequences for targeting to the peroxisomes. Also provided are isolated polypeptides encoded by such nucleotide sequences (SEQ ID NOs: 2 and 4).
Methods are provided for producing PHA in host cells. The methods involve transforming a host cell with a nucleotide molecule of the invention encoding a PHA synthase. Such host cells find use in the production of biodegradable thermoplastics. The methods additionally comprise growing the host cells for a sufficient length of time in conditions favorable for the production of PHA. The methods further involve extracting the PHA from the host cells or from the vicinity of the host cells, such as for example, a culture broth or solid medium. Preferred host cells include plant cells, bacterial cells, yeast cells, cells of non-yeast fungi, insect cells, algal cells and animal cells such as, for example, insect cells and nematode cells. The host cells of the invention can be single cells, colonies or clumps of cells, or cells within a multicellular structure or within an organism.
Methods for producing PHB in the cytosol or plastids of plants and for producing PHA in plant peroxisomes are known in the art. While the nucleotide sequences of the present invention can be used in such methods for producing PHA in plants, such methods are not known to achieve the synthesis of high levels of PHA in plants. In particular, the nucleotide sequences of the present invention find use in improved methods for producing PHA in plants, particularly in plant peroxisomes, as described in U.S. Provisional Application Serial No. 60/156,807 filed Sep. 29, 1999; herein incorporated by reference.
Methods for producing PHA in plants are provided. The methods involve genetically manipulating the genome of a plant to produce PHA. The invention encompasses plants and seeds thereof, that have been genetically manipulated to produce enzymes involved in PHA synthesis and expression cassettes containing coding sequences for such enzymes. The invention further encompasses genetically manipulated plant cells and plant tissues.
The methods for producing PHA in plants involve genetically manipulating the plant to produce at least one enzyme in the PHA biosynthetic pathway. The plants of the invention each comprise in their genomes at least one stably incorporated DNA construct, each DNA construct comprising a coding sequence for an enzyme involved in PHA synthesis operably linked to a promoter that drives the expression of a gene in a plant. Plants of the invention are genetically manipulated to produce a PHA synthase of the invention. Such PHA synthases can catalyze the synthesis of copolymers.
DNA construc

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