Polyhydroxyalkanoates (PHAs) are one class of biodegradable polymers. The first identified member of the PHAs thermoplastics was polyhydroxybutyrate (PHB), the polymeric ester of D(xe2x88x92)-3-hydroxybutyrate.
The biosynthetic pathway of PHB in the gram negative bacterium Alcaligenes eutrophus is depicted in FIG. 1. PHAs related to PHB differ in the structure of the pendant arm, R (FIG. 2). For example, R=CH3 in PHB, while R=CH2CH3 in polyhydroxyvalerate, and R=(CH2)4CH3 in polyhydroxyoctanoate.
The genes responsible for PHB synthesis in A. eutrophus have been cloned and sequenced. (Peoples et al., J. Biol. Chem., 264, 15293 (1989); Peoples et al., J. Biol. Chem. 264, 15298 (1989)). Three enzymes: xcex2-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), and PHB synthase (phbC) are involved in the conversion of acetyl-CoA to PHB. The PHB synthase gene encodes a protein of Mr=63,900 which is active when introduced into E. coli (Peoples et al., J. Biol. Chem., 26, 15298 (1989)).
Although PHB represents the archetypical form of a biodegradable thermoplastic, its physical properties preclude significant use of the homopolymer form. Pure PHB is highly crystalline and, thus, very brittle. However, unique physical properties resulting from the structural characteristics of the R groups in a PHA copolymer may result in a polymer with more desirable characteristics. These characteristics include altered crystallinity, UV weathering resistance, glass to rubber transition temperature (Tg), melting temperature of the crystalline phase, rigidity and durability (Holmes et al., EPO 00052 459; Anderson et al., Microbiol. Rev., 54, 450 (1990)). Thus, these polyesters behave as thermoplastics, with melting temperatures of 50-180xc2x0 C., which can be processed by conventional extension and molding equipment.
Traditional strategies for producing random PHA copolymers involve feeding short and long chain fatty acid monomers to bacterial cultures. However, this technology is limited by the monomer units which can be incorporated into a polymer by the endogenous PHA synthase and the expense of manufacturing PHAs by existing fermentation methods (Haywood et al., FEMS Microbiol. Lett., 57, 1 (1989); Poi et al., Int. J. Biol. Macromol., 12, 106 (1990); Steinbuchel et al., In: Novel Biomaterials from Biological Sources. D. Byron (ed.), MacMillan, N.Y. (1991); Valentin et al., Appl. Microbiol. Biotechnical, 36, 507 (1992)).
The production of diverse hydroxyacylCoA monomers for homo- and co-polymeric PHAs also occurs in some bacteria through the reduction and condensation pathway of fatty acids. This pathway employs a fatty acid synthase (FAS) which condenses malonate and acetate. The resulting xcex2-keto group undergoes three processing steps, xcex2-keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit. However, this pathway provides only a limited array of PHA monomers which vary in alkyl chain length but not in the degree of alkyl group branching, saturation, or functionalization along the acyl chain.
The biosynthesis of polyketides, such as erythromycin, is mechanistically related to formation of long-chain fatty acids. However, polyketides, in contrast to FASs, retain ketone, hydroxyl, or olefinic functions and contain methyl or ethyl side groups interspersed along an acyl chain comparable in length to that of common fatty acids. This asymmetry in structure implies that the polyketide synthase (PKS), the enzyme system responsible for formation of these molecules, although mechanistically related to a FAS, results in an end product that is structurally very different than that of a long chain fatty acid.
Because PHAs are biodegradable polymers that have the versatility to replace petrochemical-based thermoplastics, it is desirable that new, more economic methods be provided for the production of defined PHAs. Thus, what is needed are methods to produce recombinant PHA monomer synthases for the generation of PHA polymers.
The present invention provides a method of preparing a polyhydroxyalkanoate synthase. The method comprises introducing an expression cassette into a non-plant eukaryotic cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the non-plant eukaryotic cell. The DNA molecule encoding the polyhydroxyalkanoate synthase is then expressed in the cell. Thus, another embodiment of the invention provides a purified, isolated recombinant polyhydroxybutyrate synthase.
Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a eukaryotic cell. The first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the eukaryotic cell. The second expression cassette comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the eukaryotic cell. The DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer.
Another embodiment of the invention is a baculovirus expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in an insect cell.
The present invention also provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell. The nucleic acid molecule comprises a plurality of DNA segments. Thus, the nucleic acid molecule comprises at least a first and a second DNA segment. No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. The first DNA segment encodes a first module and the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate synthase.
Also provided is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module. No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. Together the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase. A preferred embodiment of the invention employs a first DNA segment derived from the vep gene cluster of Streptomyces. Another preferred embodiment of the invention employs a second DNA segment derived from the tyl gene cluster of Streptomyces.
Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing a DNA molecule into a host cell. The DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase, which synthase comprises at least a first module and a second module, is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
Also provided is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first DNA molecule and a second DNA molecule into a host cell. The first DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase. The recombinant polyhydroxyalkanoate monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first module and a second module. The first DNA molecule is operably linked to a promoter functional in a host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
Yet another embodiment of the invention is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. That is, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase and the second DNA segment encodes a module of a polyketide synthase. A preferred embodiment of the invention employs a second DNA segment encoding a module which comprises a xcex2-ketoacyl synthase amino-terminal to an acyltransferase which is amino-terminal to a ketoreductase which is amino-terminal to an acyl carrier protein which is amino-terminal to a thioesterase.
The invention also provides a method of preparing a polyhydroxyalkanoate monomer. The method comprises introducing a DNA molecule comprising a plurality of DNA segments into a host cell. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase operably linked to a promoter functional in the host cell. The second DNA segment encodes a polyketide synthase. The second DNA segment is located 3xe2x80x2 to the first DNA segment. The first DNA segment is linked to the second DNA segment so that the encoded protein is expressed as a fusion protein. The DNA molecule is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
Another embodiment of the invention is an expression cassette comprising a DNA molecule comprising a DNA segment encoding a fatty acid synthase and a polyhydroxyalkanoate synthase.
Also provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first and second module which together encode the monomer synthase.
A further embodiment of the invention is an isolated and purified DNA molecule comprising a DNA segment which encodes a Streptomyces venezuelae polyhydroxyalkanoate monomer synthase, a biologically active variant or subunit thereof. Preferably, the DNA segment encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:2. Preferably, the DNA segment comprises SEQ ID NO:1. The DNA molecules of the invention are double stranded or single stranded. A preferred embodiment of the invention is a DNA molecule that has at least about 70%, more preferably at least about 80%, and even more preferably at least about 90%, identity to the DNA segment comprising SEQ ID NO:1, e.g., a xe2x80x9cvariantxe2x80x9d DNA molecule. A variant DNA molecule of the invention can be prepared by methods well known to the art, including oligonucleotide-mediated mutagenesis. See Adelman et al., DNA, 2, 183 (1983) and Sambrook et al., Molecular Cloning: A Laboratory Manual (1989).
The invention also provides an isolated, purified polyhydroxyalkanoate monomer synthase, e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO:2, a biologically active subunit, or a biologically active variant thereof. Thus, the invention provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:2. A preferred variant polypeptide, or subunit of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 10%, more preferably at least about 50% and even more preferably at least about 90%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:2. Preferably, a variant polypeptide of the invention has one or more conservative amino acid substitutions relative to the polypeptide having the amino acid sequence comprising SEQ ID NO:2. For example, conservative substitutions include aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. The biological activity of a polypeptide of the invention can be measured by methods well known to the art.
As used herein, a xe2x80x9clinker regionxe2x80x9d is an amino acid sequence present in a multifunctional protein which is less well conserved in amino acid sequence than an amino acid sequence with catalytic activity.
As used herein, an xe2x80x9cextender unitxe2x80x9d catalytic or enzymatic domain is an acyl transferase in a module that catalyzes chain elongation by adding 2-4 carbon units to an acyl chain and is located carboxy-terminal to another acyl transferase. For example, an extender unit with methylmalonylCoA specificity adds acyl groups to a methylmalonylCoA molecule.
As used herein, a xe2x80x9cpolyhydroxyalkanoatexe2x80x9d or xe2x80x9cPHAxe2x80x9d polymer includes, but is not limited to, linked units of related, preferably heterologous, hydroxyalkanoates such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate, 3-hydroxyheptanoate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy and 5-hydroxy counterparts.
As used herein, a xe2x80x9cType I polyketide synthasexe2x80x9d is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides.
As used herein, a xe2x80x9crecombinantxe2x80x9d nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.
As used herein, a xe2x80x9cmultifunctional proteinxe2x80x9d is one where two or more enzymatic activities are present on a single polypeptide.
As used herein, a xe2x80x9cmodulexe2x80x9d is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase.
As used herein, a xe2x80x9cpremature termination productxe2x80x9d is a product which is produced by a recombinant multifunctional protein which is different than the product produced by the non-recombinant multifunctional protein. In general, the product produced by the recombinant multifunctional protein has fewer acyl groups.
As used herein, a DNA that is xe2x80x9cderived fromxe2x80x9d a gene cluster, is a DNA that has been isolated and purified in vitro from genomic DNA, or synthetically prepared on the basis of the sequence of genomic DNA.
As used herein, the terms xe2x80x9cisolated and/or purifiedxe2x80x9d refer to in vitro isolation of a DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that it can be sequenced, replicated and/or expressed. Moreover, the DNA may encode more than one recombinant Type I polyketide synthase and/or fatty acid synthase. For example, xe2x80x9can isolated DNA molecule encoding a polyhydroxyalkanoate monomer synthasexe2x80x9d is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that encode a biologically active polypeptide, fragment, or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of a polyhydroxyalkanoate monomer synthase RNA, or hybridizes to the RNA or DNA encoding the polyhydroxyalkanoate monomer synthase and remains stably bound under stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., supra.