CA2240360A1 - Metabolic engineering of polyhydroxyalkanoate monomer synthases - Google Patents

Abstract

A novel pathway for the synthesis of polyhydroxyalkanoates is provided. A method of synthesizing a recombinant polyhydroxyalkanoate monomer synthase is also provided. These recombinant polyhydroxyalkanoate synthases are derived from multifunctional fatty acid synthases or polyketide synthases and generate hydroxyacyl acids capable of polymerization by a polyhydroxyalkanoate synthase.

Description

W O 97/22711 PCT~US96/20119 METABOLIC ENGINEERING OF POLY-HYDR~XYALKANOATE MONOMER SYNTHASES

Bacl~round of the Invention Polyhydroxyalkanoates (PHAs) are one class of biodegradable polymers. The first identified mem~er of the PHAs thermoplastics was polyhydroxybutyrate (PHB), the polymeric ester of 10 D(-)-3-hydroxybutyrate.
The biosynthetic pathway of PHB in the gram negative bacterium Alcaligenes eutroph~s is depicted in Figure 1. PHAs related to PHB differ in the structure of the pendant arm, R (Figure 2).
For example, R=CH3 in PHB, while R=CH2C H 3 in 15 polyhydroxyvalerate, and R=(CH2)4CH3 in polyhydroxyoctanoate.
The genes responsible for PHB synthesis in A. etltrophus have been cloned and sequenced. (Peoples et al., J. Biol. Chem. ~
15293 (1989); Peoples et al., J. Biol. Chem. 264, 15298 (1989)). Three / enzymes~ ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), 20 and PHB synthase (phbC) are involved in the conversion of acetyl-CoA to PHB. The PHB synthase gene encodes a protein of M~63,900 which is active when introduced into E. coli (Peoples et al., J. Biol.
Chem. 264, 1529~ (1989)).
Although PHB represents the archetypical form of a 25 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 30 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., ~, 450 (1990)). Thus, these polyesters behave as thermoplastics, with melting W O 97/22711 PCT~US96/20119 temperatures of 50-180~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 5 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., FF.MS
M~crobiol. Lett., ~Z 1 (1989); Poi et al., Int. J. Biol. Macromol., 12, 106 10 (1990); Steinbuchel et al., In: Novel Biomaterials from Biolo~ical ~Qllrces. D. 13yron (ed.), MacMillan, NY (1991); Valentin et al., Appl.
Microbiol. 13iotechnical ~, 507 (1992)).
The production of diverse hydroxyacylCoA monomers for homo- and co-polymeric PHAs also occurs in some bacteria through 15 the reduction and condensation pathway of fatty acids. This pathway employs a fatty acid synthase (FAS) which condenses malonate and acetate. The resulting ,~-keto group undergoes three processing steps, ,B-keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit. However, this pathway provides only a 20 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.
25 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 30 formation of these molecules, although mechanistically related to a -WO 97/2271~ PCTAUS96/20119 FAS, results in an end product that is structurally very different than that o~ a long chain fatty acid.
Because PHAs are biodegradable po~ymers that have the versatility to replace petrochemical-based thermoplastics, it is 5 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.

SumrnAry of the Invention 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 15 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 puri~ied, isolated recombinant polyhydroxybutyrate synthase.
Another embodiment of the invention is a method of preyaring a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a eukaryotic cell. The ~irst expression cassette comprises a DNA
se~nent encoding a fatty acid synthase in which the dehydrase 25 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 linlced to a promoter functional in the eukaryotic cell. The DNA segments in the expression cassettes are expressed in the cell so 30 as to yield a polyhydroxyalkanoate polymer.

W O 97/22711 PCT~US96/20119 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 Saccharopolysporn 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 WO 97t22711 PCTAUS96J20119 polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The DNA encoding the recomb;nant polyhydroxyalkanoate monomer synthase, which synthase comprises at least a first module and a second module, is 5 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 10 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 15 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 20 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 2~ least a Rrst 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 ,B-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 5 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 segrnent. 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
10 segment is located 3' 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 20 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 25 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 strepto~flyces venezuelae polyhydroxyalkanoate monomer synthase, a biologically active variant or subunit thereof. Preferably, the DNA
30 segment encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:2. Preferably, the DNA segment comprises W O 971227~1 PCTfUS96~0tl9 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 5 90%, identity to the DNA segment comprising SEQ ID NO:1, e.g., a "variant" DNA molecule. ~ 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, ~, 183 (1983) and Sambrook et al., Molecular Cloning: A Laboratory 10 ~anual ~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 15 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 am;no acid se~uence comprising SE~ ID NO:2. A preferred variant polypeptide, or subunit of a polypeptide, of the invention includes a variant or subunit 20 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 25 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 30 hydrophilic amino acids. The biological activity of a polypeptide of the invention can be measured by methods well known to the art.

W O 97/22711 PCT~US96~0119 As used herein, a "linker region" 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 "extender unit" 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 "polyhydroxyalkanoate" or "PHA"
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 "Type I polyketide synthase" 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 "recombinant" nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified i~ 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 "multifunctional protein" is one where two or more enzymatic activities are present on a single polypeptide.

W O 97~2711 P~US96~I19 As used herein, a "module" 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 "premature termination product" is a 5 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 "derived from" a gene 10 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 "isolated and/or purified" refer to in ~itro isolation of a DNA or polypeptide molecule from its 15 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, "an isolated DNA molecule 20 encoding a polyhydroxyalkanoate monomer synthase " is RNA or DNA containing greater than 7, ~releldbly 15, and more ~l-efeLably 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 25 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., supr~.
3û

W O 97~2711 PCT~US96/20119 Brief Descxiption of the Figures Figure 1: The PHB biosynthetic pathway in A.
eu tro phus .
Figure 2: Molecular structure of common bacterial PHAs. Most of the known PHAs are polymers of 3-hydroxy acids possessing the general formula shown. For example, R=CH3 in PHB, R=CH2CH3 in polyhydroxyvalerate (PHV), and R=(CH2)4CH3 in polyhydroxyoctanoate (PHO).
Figure 3: Comparison of the natural and recombinant pathways for PHB synthesis. The three enzymatic steps of PHB synthesis in bacteria involving 3-ketothiolase, acetoacetyl-CoA
reductase, and PHB synthase are shown on the left. The two enzymatic steps involved in PHB synthesis in the pathway in S121 cells containing a rat fatty acid synthase with an inactivated dehydrase domain (ratFAS206) are shown on the right.
Figure 4: Schematic diagram of the molecular organization of the tyl polyketide synthase (PKS) gene cluster. Open arrows correspond to individual open reading frames (ORFs) and numbers above an ORF denote a multifunctional module or synthase unit (SU) . AT=acyltransferase; ACP=acyl carrier protein; KS= ,~-ketoacyl synthasei KR=ketoreductase; D~=dehydrase; ER=enoyl reductase; TE=thioesterase; MM=methylmalonylCoA; M=malonyl CoA; EM=ethylmalonyl CoA. Module 7 in tyl is also known as Module F.
Figure 5: Schematic diagram of the molecular organization of the met PKS gene cluster.
Figure 6: Strategy for producing a recombinant PHA
monomer synthase by domain replacement.
Figure 7: (A) 10% SDS-PAGE gel showing samples from various stages of the purification of P~A synthase; lane 1, W O 97/22711 PCTAUS96~0119 molecular weight markers; lane 2, total protein of uninfected insect cells; lane 3, total protein of insect cells expressing a rat FAS ( 200 kDa;
Joshi et al., Bioche~ J. ~, 143 (1993)); lane 4, total protein of insect cells expressing PHA synthase; lane 5, soluble protein from sample in 5 lane 4; lane 6, pooled hydroxylapatite (HA) fractions containing PHA
synthase. (B) Western analysis of an identical gel using rabbit~ PHA
synthase antibody as probe. Bands designated with arrows are: a, intact PHB synthase with N-terminal alanine at residue 7 and serine at residue 10 (A7/S10); b, 44 kDa fragment of PE~B synthase with N-10 terminal alanine at residue 181 and asparagine at residue 185(A181 /N185); c, PHB synthase fragment of approximately 30 kDa apparently blocked based on resistance to Edman degradation; d, 22 kDa fragment with N-terminal glycine at residue 187 (G187). Band d apparently does not react with rabbit-oc-PHB synthase antibody (B, lane 15 6). The band of similar size in B, lane 4 was not further identified.
' Figure 8: N-terminal analysis of PHA synthase purified from insect cells. (a) The expected N-terminal 25 amino acid sequence of A. eutrophus PHA synthase. (b&c) The two N-terminal sequences determined for the A. eu~rophus PHA synthase produced 20 in insect cells. The bolded sequences are the actual N-termini determined.
Figure 9: Spectrophotometric scans of substrate, 3-hydroxybutyrate CoA (HBCoA) and product, CoA. The wavelength at which the direct spectrophotometric assays were carried out (232 nm~
25 is denoted by the arrow; substrate, HBCoA (- ) and product, CoA (o).
Figure 10: Velocity of the hydrolysis of HBCoA as a function of substrate concentration. Assays were carried out in 40 or 200 ,ul assay volumes with enzyme concentration remaining constant at 0.95 mg/ml (3.8 ,ug/40111 assay). Velocities were calculated from the 30 linear portions of the assay curves subsequent to the characteristic lag W O 97/22711 PCT~US96/20119 period. The substrate concentration at half-optimal velocity, the apparent l~m value, was estimated to be 2.5 mM from this data.
Figure 11: Double reciprocal plot of velocity versus substrate concentration. The concave upward shape of this plot is similar to results obtained by Fukui et al. (Arch. Microbiol. 110, 149 (1976)) with granular PHA synthase from Z. ramigera.
Figure 12: Velocity of the hydrolysis of HBCoA as a function of enzyme concentration. Assays were carried out in 40 ~Ll assay volumes with the concentration HBCoA remaining constant at 8~M.
Figure 13: Specific activity of PHA synthase as a function of enzyme concentration.
Figure 14: pH activity curve for soluble P~A synthase produced using the baculovirus ~y~L~ . Reactions were carried out in the presence of 200 mM Pi. Buffers of pH<10 were prepared with potassium phosphate, while buffers of pH>10 were prepared with the appropriate proportion of Na3PO4.
Figure 15: Assays of the hydrolysis of HBCoA with varying amounts of PHA synthase. Assays were carried out in 40 ,ul assay volumes with the concentration of HBCoA remaining constant at 8 tlM. Initial A232 values, originally between 0.62 and 0.77, were normalized to 0.70. Enzyme amounts used in these assays were, from the upper-most curve, 0.38, 0.76, 1.14, 1.52, 1.90, 2.28, 2.66, 3.02, 3.42, 7.6, and 15.2,ug, respectively.
Figure 16: SDS/PAGE analysis of proteins synthesized at various time-points during infection of Sf21 cells. Approximately 0.5 mg of total cellular protein from various samples was fractionated on a 10% polyacrylamide gel. Samples include: ur-infected cells, lanes 1-4, days 0, 1, 2, 3 respectively; infection with BacPAK6::phbC alone, CA 02240360 1998-06-ll WO 97~22711 PCT~US96J20119 lanes 5-8, days 0, 1, 2, 3 respectively; infection with baculoviral clone containing ratFAS206 alone, lanes 9-12, days 0,1, 2, 3 respectively; and ratFAS206 and BacPAK6 infected cells, lanes 13-16 days 0, 1, 2, 3, respectively. A=mobility of FAS, B=mobility of PHA synthase.
5 Molecular weight standard lanes are marked M.
Figure 17: Gas chromatographic evidence for PHB
accumulation in S.f21 cells. Gas chromatograms from various samples are superimposed. PHB standard (Sigma) is chromatogram ~7 showing a propylhydroxybutyrate elution time of 10.043 minutes 10 (s, arrow). The gas chromatograms of extracts of the uninfected (#1);
singly infected with ratFAS206 (~2, day 3); and singly infected with PHA synthase (~3, day 3) are shown at the bottom of the figure. Gas chromatograms of extracts of dual-infected cells at day 1 (~4), 2 (#5), and 3 (~6) are also shown exhibiting a peak eluting at 10.096 minutes 15 (x, arrow). The peak of dual-infected, day 3 extract (#6) was used for mass spectrometry (MS) analysis.
Figure 18: Gas-chromatography-mass spectrometry analysis of PHB. The characteristic fragmentation of propylhydroxybutyrate at m/z of 43, 60, 87, and 131 is shown. A) 20 standard PHB from bacteria (Sigma), and B) peak X from ratFAS206 and BacPAK6: phbC baculovirus infected, day 3 ($~6, Figure 17) Sf21 cells expressing rat FAS dehydrase inactivated protein and PHA
synthase.
Figure 19: Map of the vep (Streptomyces venezuelae 25 polyene encoding) gene cluster.
Figure 20: Plasmid map of pDHS502.
Figure 21: Plasmid map of pDHS505.
~ Figure 22: Cloning protocol for pDHS505.
Figure 23: Nucleotide sequence (S~Q ID NO:1) and 30 corresponding amino acid sequence (SEQ ID NO:22) of the vep ORFI.

W O 97/22711 PCT~US96/~0119 Detailed ~escription of the Invention The invention described herein can be used for the production of a diverse range of biodegradable PHA polymers through genetic redesign of DNA encoding a FAS or Streptomyces 5 spp. Type I PKS polypeptide to provide a recombinant PHA monomer synthase. Different PHA synthases can then be tested for their ability to polymerize the monomers produced by the recombinant PHA
synthase into a biodegradable polymer. The invention also provides a method by which various PHA synthases can be tested for their 1() specificity with respect to different monomer substrates.
The potential uses and applications of PHAs produced by PHA monomer synthases and PHA synthases includes both medical and industrial applications. Medical applications of PHAs include surgical pins, sutures, staples, swabs, wound dressings, blood vessel 15 replacements, bone replacements and plates, stimulation of bone growth by piezoelectric properties, and biodegradable carrier for long-term dosage of pharmaceuticals. Industrial applications of PHAs include disposable items such as baby diapers, packaging containers, bottles, wrappings, bags, and films, and biodegradable carriers for long-20 term dosage of herbicides, fungicides, insecticides, or fertilizers.
In animals, the biosynthesis of fatty acids de ~ovo frommalonyl-CoA is catalyzed by FAS. For example, the rat FAS is a homodimer with a subunit structure consisting of 2505 amino acid residues having a molecular weight of 272,340 Da. Each subunit 25 consists of seven catalytic activities in separate physical domains (Amy et al., Proc. Natl. Acad. Sci. USA, ~, 3114 (1989)). The physical location of six of the catalytic activities, ketoacyl synthase (KS), malonyl/acetyltransferase (M/AT), enoyl reductase (ER), ketoreductase (KR), acyl carrier protein (ACP), and thioesterase (TE), 30 has been established by (1) the identification of the various active site residues within the overall amino acid sequence by isolation of CA 02240360 1998-06-ll WO 97/227'11 PCT~US96~0119 catalytically active fragments from limited proteolytic digests of the whole FAS, (2) the identification of regions within the FAS that exhibit sequencc similarity with various monofunctional proteins, (3) expression of DNA encoding an amino acid sequence with catalytic 5 activity to produce recombinant proteins, and (4) the identification of DNA that does not encode catalytic activity, i.e., DNA encoding a linker region. (Smith et al., Proc. Natl. Acad. Sci. USA, Z~, 1184 (1976);
Tsukamoto et al., J. Biol. Chem., 263 16225 (1988); Rangan et al., L
Biol. Chem. ~~~, 1918û (1991)).
The seventh catalytic activity, dehydrase (DH), was identified as physically residing between AT and ER by an amino acid comparison of FAS with the amino acid sequences encoded by the three open reading frames of the eryA polyl~etide synthase (PKS) gene cluster of Saccharopolyspor~ erythraea. The three polypeptides that 15 comprise this PKS are constructed from "modules" which resemble animal FAS, both in terms of their amino acid sequence and in the ordering of the constituent domains (Donadio et al., Gene, 111, 51 (1992); Benh et al., Eur. T. Biochem. 204 39 (1992)).
One embodiment of the invention employs a FAS in 20 which the DH is inactivated (FAS DH-). The FAS DH- employed in this embodiment of the invention is preferably a eukaryotic FAS DH-and, more preferably, a mammalian FAS DH-. The most preferred embodiment of the invention is a FAS where the active site in the DH has been inactivated by mutation. For example, Joshi et al. (L
25 ~iol. Chem., 268, 22508 (1993)) changed the His878 residue in the rat ~ FAS to an alanine residue by site directed mutagenesis. In vitro studies showed that a FAS with this change (ratFAS206) produced 3-hydroxybutyrylCoA as a premature termination product from acetyl-CoA, malonyl-CoA and NADPH.

W O 97/22711 PCT~US96/20119 As shown below, a FAS DH- effectively replaces the ,B-ketothiolase and acetoacetyl-CoA reductase activities of the natural pathway by producing D(-)-3-hydroxybutyrate as a premature termination product, rather than the usual 16-carbon product, palmitic acid. This premature termination product can then be incorporated into PHB by a PHB s~nthase (See Example 2).
Another embodiment of the invention employs a recombinant Streptomyces spp. PKS to produce a variety of ~-hydroxyCoA esters that can serve as monomers for a PHA synthase.
One example of a DNA encoding a Type I PKS is the eryA gene cluster, which governs the synthesis of erythromycin aglycone deoxyerythronolide B (DEB). The gene cluster encodes six repeated units, termed modules or synthase units (SUs). Each module or SU, which comprises a series of putative FAS-like activities, is responsible for one of the six elongation cycles required for DEB formation. Thus, - the processive synthesis of asymmetric acyl chains found in complex polyketides is accomplished through the use of a programmed protein template, where the nature of the chemical reactions occurring at each point is determined by the specificities in each SU.
Two other Type I PKS are encoded by the tyl (tylosin) (Figure 4) and met (methymycin) (Figure 5) gene clusters. The macrolide multifunctional synthases encoded by tyl and met provide a greater degree of metabolic diversity than that found in the eryA
gene cluster. ~he PKSs encoded by the eryA gene cluster only catalyze chain elongation with methylmalonylCoA, as opposed to tyl and m e t PKSs, which catalyze chain elongation with malonylCoA, methylmalonylCoA and ethylmalonylCoA. Specifically, the tyl PKS
includes two malonylCoA extender units and one ethylmalonylCoA
extender unit, and the met PKS includes one malonylCoA extender unit. Thus, a preferred embodiment of the invention includes, but is CA 02240360 l998-06-ll W O 97/22711 PCTJUS96~119 not limited to, replacing catalytic activities encoded in met PKS open readimg frame 1 (ORF1) to provide a DNA encoding a protein that possesses the required keto group processing capacity and short chain acylCoA ester starter and extender unit specificity necess~ry to provide a saturated ,~-hydroxyhexanoylCoA or unsaturated ,~-hydroxyhexenoylCoA monomer.
In order to manipulate the catalytic specificities within each module, DNA encoding a catalytic activity must remain undisturbed. To identify the amino acid sequences between the amino acid sequences with catalytic activity, the "linker regions,"
amino acid sequences of related modules, preferably those encoded by more than one gene cluster, are compared. Linker regions are amino acid sequences which are less well conserved than amino acid sequences with catalytic activity. Witkowski et al., Eur. J. Biochem., ~ , 571 (1991).
In an alternative embodiment of the invention, to provide a DNA encoding a Type I PKS module with a TE and lacking a functional DI3, a DNA encoding a module F, containing KS, MT, KR, ACP, and TE catalytic activities, is introduced at the 3' end of a 2û DNA encoding a first module (Figure 6). Module 1~ introduces the final (R)-3-hydroxyl acyl group at the final step of PHA monomer synthesis, as a result of the presence of a TE domain. DNA encoding a module F is not present in the eryA PKS gene cluster (Donadio et al., sllpra, 1991).
A DNA encoding a recombinant monomer synthase is inserted into an expression vector. The expression vector employed varies depending on the host cell to be transformed with the expression vector. That is, vectors are employed with transcription, translation and/or post-translational signals, such as targeting signals, necessary for efficient expression of the genes in various host cells W O 97/22711 PCTrUS96/20119 into which the vectors are introduced. Such vectors are constructed and transformed into host cells by methods well known in the art.
See Sambrook et al., Molecular Cloning: ~ Laboratory Manual, Cold Spring Harbor (1989). Preferred host cells for the vectors of the 5 invention include insect, bacterial, and plant cells. Preferred insect cells include Spodoptera frugiperda cells such as Sf21, and Tric7loplusia 7zi cells. Preferred bacterial cells include Escherichia coli, Streptomyces and Pse1ldomon~s. Preferred plant cells include monocot and dicot cells, such as maize, rice, wheat, tobacco, legumes, 10 carrot, squash, canola, soybean, potato, and the like.
Moreover, the appropriate subcellular compartment in which to locate the enzyme in eukaryotic cells must be considered when constructing eukaryotic expression vectors. Two factors are important: the site of production of the acetyl-CoA substrate, and the 15 available space for storage of the P~IA polymer. To direct the enzyme to a particular subcellular location, targeting se~uences may be added to the sequences encoding the recombinant molecules.
The baculovirus system is particularly amenable to the introduction of DNA encoding a recombinant FAS or a PKS
20 monomer synthase because an increasing variety of transfer plasmids are becoming available which can accommodate a large insert, and the virus can be propagated to high titers. Moreover, insect cells are adapted readily to suspension culture, facilitating relatively large scale recombinant protein production. Further, recombinant proteins tend 25 to be produced exclusively as soluble proteins in insect cells, thus, obviating the need for refolding, a task that might be particularly daunting in the case of a large multifunctional protein. The Sf21/baculovirus system has routinely expressed milligram ~uantities of catalytically active recombinant fatty acid synthase. Finally, the 30 baculovirus/insect cell system provides the ability to construct and WO 97/22711 PCTAUS96~20~ 19 analyze different synthase proteins for the ability to polymerize monomers into unique biodegradable polymers.
A further embodiment of the invention is the introduction of at least one DNA encoding a PHA synthase and a 5 DNA encoding a PHA monomer synthase into a host cell. Such synthases include, but are not limited to, A. e1ltroph~s 3-hydroxy, 4-hydroxy, and 5-hydroxy alkanoate synthases, Rhodococcus ruber C3-Cs hydroxyalkanoate synthases, Pseudomonas oleororans C6-C14 hydroxyalkanoate synthases, P. putida C6-C14 hydroxyalkanoate 10 synthases, P. aeruginosa Cs-Clo hydroxyalkanoate synthases, P.
resinovorans C4-Clo hydroxyalkanoate synthases, Rhodospirillum rubr~m C4-C7 hydroxyalkanoate synthases, R. gelatlnorus C4-C7, ~hiocapsn pfennigii C4-C8 hydroxyalkanoate synthases, and Bacillus megaterium C4-C5 hydroxyalkanoate synthases.
The introduction of DNA(s) encoding more than one PHA synthase may be necessary to produce a particular PHA polymer due to the specificities exhibited by different PHA synthases. As multifunctional proteins are altered to produce unusual monomeric structures, synthase specificity may be problematic for particular 20 substrates. Although the A. eutrophus PHB synthase utilizes only C4 and C5 compounds as substrates, it appears to be a good prototype synthase for initial studies since it is known to be capable of producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (Kunioka et al., Macromolecules ~, 694 (1989)) as well as copolymers of 3-25 hydroxyvalerate, 3-hydroxybutyrate, and 5-hydroxyvalerate (Doi et al., ~ Macromolecules 19 2860 (1986)). Other synthases, especially those of Pseudomonas aeruginosa (Timm et al., Eur. J. Biochem., 209, 15 (1992)) and Rhodococcus ruber (Pieper et al., FEMS Microbiol. Lett.
~, 73 (1992)), can also be employed in the practice of the invention.
30 Synthase specificity may be alterable through molecular biological methods.

CA 02240360 l998-06-ll W O 97/22711 PCT~US96/20119 In yet another embodiment of the invention, a DNA
encoding a FAS and a PHA synthase can be introduced into a single expression vector, obviating the need to introduce the genes into a host cell individually.
A further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS
module P. Module F will carry out the final chain extension to include two additional carbons and the reduction of the ,B-keto group, 1~ which results in a (R)-3-hydroxy acyl CoA moiety.
To produce this recombinant protein, DNA encoding the FAS TE is replaced with a DNA encoding a linker region which is normally found in the ACP-KS interdomain region of bimodular ORFs. DNA encoding a module F is then inserted 3' to the DNA
15 encoding the linker region. Different linker regions, such as those described below, which vary in length and amino acid composition, can be tested to determine which linker most efficiently mediates or allows the required transfer of the nascent saturated fatty acid intermediate to module F for the final chain elongation and keto 20 reduction steps. The resulting DNA encoding the protein can then be tested for expression of long chain ,13-hydroxy fatty acids in insect cells, such as S.f21 cells, or S~reptomyces, or Pseudomonas. The expected 3-hydroxy C-18 fatty acid can serve as a potential substrate for PHA
synthases which are able to accept long chain alkyl groups. A
25 preferred embodiment of the invention is a FAS that has a chain length specificity between 4-22 carbons.
Examples of linker regions that can be employed in this embodiment of the invention include, but are not limited to, the ACP-KS linker regions encoded by the tyl ORFI ~ACPl-KS2; ACP2-wo s7n27ll PCT~US96~20119 KS3), and ORF3 (ACPs-KS6), and eryA OR;FI (ACPl-KSl; ACP2-KS2), 0RF2 (ACP3-KS4) and ORF3 (ACPs-KS6).
This approach can also be used to produce shorter chain fatty acid groups by limiting the ability of the FAS unit to generate 5 long chain fatty acids. Mutagenesis of DNA encoding various FAS
catalytic activities, starting with the KS, may result in the synthesis of short chain (R)-3-hydroxy fatty acids.
The PHA polymers are then recovered from the biomass.
Large scale solvent extraction can be used, but is expensive. An 10 alternative method involving heat shock with subsequent enzymatic and detergent digestive processes is also available ~Byron, Trends Biotechnical ~, 246 (1987); Holmes, In: Developments in Crystalline Polymers D.C. Bassett (ed), pp. 1-65 ~1988)). PHB and other PHAs are read;ly extracted from microorganisms by chlorinated hydrocarbons.
15 Refluxing with chloroform has been extensively used; the resulting solution is filtered to remove debris and concentrated, and the polymer is precipitated with methanol or ethanol, leaving low-molecular-weight lipids in solution. Longer-side-chain PHAs show a less restricted solubility than PHB and are, for example, soluble in 20 acetone. Other strategies adopted include the use of ethylene carbonate and propylene carbonate as disclosed by Lafferty et al.
(Chem. Rundschau, ~Q 14 (1977)) to extract PHB from biomass.
Scandola et al., (Int. J. Biol. Microbiol. ~1373 (1988)) reported that 1 M
HCl-chloroform extraction of Rhizobium meliloti yielded PHB of ~v 25 = 6 x 104 compared with 1.4 x 106 when acetone was used.
- Methods are well known in the art for the determination of the PHB or PH~ content of microorganisms, the composition of PHAs, and the distribution of the monomer units in the polymer.
Gas chromatography and high-pressure liquid chromatography are 30 widely used for quantitative PHB analysis. See Anderson et al.
Microbiol. l~ev., ~, 450 ~1990) for a review of such methods. NMR

W O 97/22711 PCT~US96/20119 techniques can also be used to determine polymer composition, and the distribution of monomer units.
The invention has been described with reference to various specific and preferred embodiments and will be further 5 described by reference to the following detailed examples. It is understood however, that there are many extensive variations, and modifications on the basic theme of the present invention beyond that shown in the examples and description, which are within the spirit and scope of the present invention.
I. Experimental Procedures Materials and Methods 15 Materials. Sodium R-(-)-3-hydroxybutyrate, coenzyme-A, ethylchloroformate, pyridine and diethyl ether were purchased from Sigma Chemical Co. Amberlite IR-120 was purchased from Mallinckrodt Inc. 6-O-(N-Heptylcarbamoyl)methyl a- D -glucopyranoside (Hecameg) was obtained from Vegatec (Villeejuif, France). Two-piece spectrophotometer cells with pathlengths of 0.1 (#20/0-Q-1) and 0.01 cm (~20/0-Q-0.1) were obtained from Starna Cells Inc., (Atascadero, CA). Rabbit anti-A. eutrophus PHA synthase antibody was a gracious gift from Dr. F. Srienc and S. Stoup (Biological Process Technology Institute, University of Minnesota). Sf~1 cells and ~. 7li cells were kindly provided by Greg Franzen (R&D Systems, Minneapolis, MN) and Stephen Harsch (Department of Veterinary Pathobiology, University of Minnesota), respectively.
Plasmid pFAS206 and a recombinant baculoviral clone encoding FAS206 (Joshi et al., J. Biol. Chem., 268 22508 (1993)) were - 30 generous gifts of A. Joshi and S. Smith. Plasmid pAet~1 (Peoples et al., WO g7/22711 PC'r./US96J20~9 J. Biol. ~hem., ~~~, 15298, ~1989)), the source of the A. eutrophus PHBsynthase, was obtained from A. Sinskey. Baculovirus transfer vector, pBacPAK9, and linearized baculoviral DNA, were obtained from Clontech Inc. (Palo Alto, CA). Restriction enzymes, T4 DNA ligase, E.
5 coli DH5a competent cells, molecular weight standards, lipofectin reagent, Grace's insect cell medium, fetal bovine serum (FBS), and antibiotic/antimycotic reagent were obtained from GIBCO-BRL
(Grand Island, NY). Tissue culture dishes were obtained from Corning Inc. Spinner flasks were obtained from Bellco Glass Inc.
10 Seaplaque agarose GTG was obtained from FMC Bioproducts Inc.

Methods.
Preparation of R-3HBCoA. R-(-)-3 HBCoA was prepared by the mixed anhydride method described by Haywood et al., FEMS
15 Microbiol. Lett., ~, 1 (1989). 60 mg (0.58 mmol) of R-(-)-3 hydroxybutyric acid was freeze dried and added to a solution of 72 mg of pyridine in 10 ml diethyl ether at 0~C. Ethylchloroformate (100 mg) was added, and the mixture was allowed to stand at 4~C for 60 minutes. Insoluble pyridine hydrochloride was removed by 20 centrifugation. The resulting anhydride was added, dropwise with mixing, to a solution of 100 mg coenzyme-A (0.13 mmol) in 4 ml 0.2 M potassium bicarbonate, pH 8.0 at 0~C. The reaction was monitored by the nitroprusside test of Stadtman, Meth. Enzymol. ~ 931 (1957), to ensure sufficient anhydride was added to esterify all the coenzyme-A.
25 The concentration of R-3-HBCoA was determined by measuring the absorbance at 260 nm (e = 16.8 mM-l cm-l; 18).
Construction of pBP-phbC. The phbC gene (approximately 1.8 kb) was excised from pAet41 (Peoples et al., J. Biol. Chem., 264, 15293 (1989)) by digestion with BstBI and StuI, purified as described by - 30 Williams et al. (Gçne, 109 4~5 (1991)), and ligated to pBacPAK9 W O 97/22711 PCT~US96/20119 digested with Bs~BI and StuI. This resulted in pBP-phbC, the baculovirus transfer vector used in formation of recombinant baculovirus particles carrying phbC.
Large scale expression of PHA synthase. A 1 L culture of T.
5 ni cells (1.2x106 cells/ml) in logarithmic growth was infected by the addition of 50 ml recombinant viral stock solution ( 2.5x108 pfu/ml) resulting in a multiplicity of infection (MOI) of 10. This infected culture was split between two Bellco spinners (350 ml/500 ml spinner, 700 ml/1 L spinner) to facilitate oxygenation of the culture. These 10 cultures were incubated at 28~C and stirred at 60 rpm for 60 hours.
Infected cells were harvested by centrifugation at 1000 x g for 10 minutes at 4~C. Cells were flash-frozen in liquid N2 and stored in 4 equal aliquots, at -80~C until purification.
Insect cell maintenance and recombinant baculovirus 15 formation. Sf21 cells were maintained at 26-28~C in Grace's insect cell medium supplemented with 10% FBS, 1.0% pluronic F68, and 1.0%
antibiotic/antimycotic (GIBCO-BRL). Cells were typically maintained in suspension at 0.2 - 2.0 x 106/ml in 60 ml total culture volume in 100 ml spinner flasks at 55-65 rpm. Cell viability during the culture 20 period was typically 95-100%. The procedures for use of the transfer vector and baculovirus were essentially those described by the manufacturer (Clontech, Inc.). Purified pBP-phbC and linearized baculovirus DNA were used for cotransfection of S.~1 cells using the liposome mediated method (Felgner et al., Proc. Natl. Acad. Sci. US~, 25 ~L 7413 (1987)) utilizing Lipofectin (GIBCO-BRL). Four days later cotransfection supernatants were utilized for plaque purification.
Recombinant viral clones were purified from plaque assay plates containing 1.5% Seaplaque GTG after 5-7 days at 28~C. Recombinant viral clone stocks were then amplified in T25-f~ask cultures (4 ml, 30 3x106/ml on day 0) for 4 days; infected cells were determined by their morphology and size and then screened by SDS/PAGE using 10%

W O 97/22711 PC~MS96~20~19 polyacrylamide gels (Laemmli, Nature 227 680 (1970)) for production of PHA synthase.
Purification of PHA synthase from BTI-TN-5B1-4 T ni cells. Purification of PHA synthase was performed according to the method of Gerngross et al., ~iochemistry ~ 9311 (1994) with the following alterations. One aliquot ( 110 mg protein) of frozen cells was thawed on ice and resuspended in 10 mM KPi (pH 72), 5%
glycerol, and 0.05% Hecameg (Buffer A) containing the following protease inhibitors at the indicated final concentrations: benzamidine (2mM), phenylmethylsulfonyl fluoride (PMSF, 0.4 mM), pepstatin (2 mg/ml), leupeptin (2.5 mg/ml), and Na-p-tosyl-l-lysine chloromethyl ketone (TLCK, 2 mM). EDTA was omitted at this stage due to its incompatibility with hydroxylapatite (HA). This mixture was homogenized with three series of 10 strokes each in two Thomas homogenizers while partially submerged in an ice bath and then sonicated for 2 minutes in a Branson Sonifier 250 at 30% cycle, 30%
power while on ice. All subsequent procedures were carried out at 4~C.
The lysate was immediately centrifuged at 100000 x g in a Beckrllan 50.2Ti rotor for 80 minutes, and the resulting supernatant (10.5 ml, 47 mg) was immediately filtered through a 0.45 mm Uniflow filter (Schleicher and Schuell Inc., Keene, N.H.) to remove any remaining insoluble matter. Aliquots of the soluble fraction (1.5 ml, 7 mg) were loaded onto a 5 ml BioRad Econo-Pac HTP column that had lbeen equilibrated with Buffer A (+ protease inhibitor mix) attached to a BioRad Econo-system, and the column was washed with 30 ml Buffer A. All chromatographic steps were carried out at a flow rate of 0.8 ml/rninute. PHA synthase was eluted from the HA column with a 32 x 32 ml linear gradient from 10 to 300 mM KPi.
Fraction collection tubes were prepared by addition of 30 ml of 100 mM EDTA to provide a metalloprotease inhibitor at 1 mM

W O 97/22711 PCT~US96/20119 immediately after HA chromatography. PHA synthase was eluted in a broad peak between 110-180 mM KPi. Fractions (3 ml) containing significant PHA synthase activity were pooled and stored at 0~C until the entire soluble fraction had been run through the chromatographic 5 process. Pooled fractions then were concentrated at 4~C by use of a Centriprep-30 concentrator (Amicon) to 3.8 mg/ml. Aliquots (0.5 ml) were either flash-frozen and stored in liquid N2 or glycero} was added to a final concentration of 50% and samples (1.9 mg/ml) were stored at -20~C.
Western analysis. Samples of T. ni cells were fractionated by SDS-PAGE on 10% polyacrylamide gels, and the proteins then were transferred to 0.2 mm nitrocellulose membranes using a BioRad Transblot SD Semi-Dry electrophoretic transfer cell according to the manufacturer. Proteins were transferred for 1 hour at 15 V. The 15 membrane was rinsed with doubly distilled H2O, dried, and treated with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tween) and 3% nonfat dry milk to block non-specific binding sites. Primary antibody (rabbit anti-PHA synthase) was applied in fresh blocking solution and incubated at 25~C for 2 hours. Membranes were 20 then washed four times for 10 minutes with PBS-Tween followed by the addition of horseradish peroxidase-conjugated goat-anti-rabbit antibody (Boehringer-Mannheim) diluted lO,OOOX in fresh blocking solution and incubated at 25~C for 1 hour. Membranes were washed finally in three changes (10 minutes) of PBS, and the immobilized 25 peroxidase label was detected using the chemiluminescent LumiGLO
substrate kit (Kirkegaard and Perry, Galthersburg, MD) and X-ray film.
N-terminal analysis. Approximately 10 mg of purified PHA synthase was run on a 10% SDS-polyacrylamide gel, transferred to PVDF (Immobilon-PSQ, Millipore Corporation, Bedford, MA), 30 stained with Amido Black, and sequenced on a 494 Procise Protein WO 97/22711 PCT/US96~201 19 Sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, California).
Double-infection protocol. Four 100 ml spinner flasks were each inoculated with 8 x 107 cells in 50 ml of fresh insect 5 medium. To flask 1, an additional 20 ml of fresh insect medium was added (uninfected control); to flask 2, 10 ml BacPAK6::phbC viral stock ( 1 x 108 pfu/ml) and 10 ml fresh insect medium were added; to flask 3, 10 ml BacPAK6::FAS206 viral stock ( 1 x 10 8 pfu/ml) and 10 ml fresh insect medium were added; and to flask 4, 10 ml 10 BacPAK6::p~tbC viral stock ( 1 x 10 8 pfu/ml) and 10 ml BacPAK6::FAS206 viral stock ( 1 x 108 pfu/ml) were added. These viral infections were carried out at a multiplicity of infection of approximately 10. Cultures were maintained under normal growth conditions and 15 ml samples were removed at 24, 48, and 72 hour 15 time points. Cells were collected by gentle centrifugation at 1000 x g for ~ minutes, the medium was discarded, and the cells were immediately stored at -70~C.
PHA synthase assays. Coenzyme A released by PHA
synthase in the process of polymerization was monitored precisely as 20 described by Gerngross et al. (supra) using 5,5'-dithiobis (2-nitrobenzoic acid, DTNB) (Ellman, Arch. Biochem. 13iophys. ~, 70 (1959))-The presence of HBCoA was monitored spectrophotometrically. Assays were performed at 25~C in a Hewlett 25 Paclcard 8452A diode array spectrophotometer equipped with a water jacketed cell holder. Two-piece Starna Spectrosil spectrophotometer cells with pathlengths of 0.1 and 0.01 cm were employed to avoid errors arising from the compression of the absorbance scale at higher values. Absorbance was monitored at 232 nm, and Ez32nm of 4.5 X 103 30 M l cm-l was used in calculations. One unit (U) of enzyme is the amount required to hydrolyze 1 mmol of substrate minute-l. Buffer CA 02240360 l998-06-ll (0.15 M KPi, pH 7.2) and substrate were equilibrated to 25~C and then combined in an Eppendorf tube also at 25~C. Enzyme was added and mixed once in the pipet tip used to transfer the entire mixture to the spectrophotometer cell. The two piece cell was immediately 5 assembled, placed in the spectrophotometer with the cell holder (type CH) adapted for the standard 10 mm path length cell holder of the spectrophotometer. Manipulations of sample, from mixing to initiation of monitoring, took only 10-15 seconds. Absorbance was continually monitored for up to 10 minutes. Calibration of reactions 10 was against a solution of buffer and enzyme (no substrate) which lead to absorbance values that represented substrate only.
PHB assay. PHB was assayed from Sf21 cell samples according to the propanolysis method of Riis et al., J. Chromo., 445, 285 (1988). Cell pellets were thawed on ice, resuspended in 1 ml cold 15 ddH2O and transferred to 5 ml screwtop test tubes with teflon seals. 2 ml ddH2O was added, the cells were washed and centrifuged and then 3 ml of acetone were added and the cells washed and centrifuged.
The samples were then dessicated by placing them in a 94~C oven for 12 hours. The following day 0.5 ml of 1,2-dichloroethane, 0.5 ml 20 acidified propanol (20 ml HCl, 80 ml l-propanol) and 50 ml benzoic acid standard were added and the sealed tubes were heated to 100~C in a boiling water bath for 2 hours with periodic vortexing. The tubes were cooled to room temperature and the organic phase was used for gas-chromatographic (GC) analysis using a Hewlett Packard 5890A gas-25 chromatograph equipped with a Hewlett Packard 7673A automaticinjector and a fused silica capillary column, DB-WAX 30W of 30 meter length. Positive samples were further subjected to GC-mass spectrometric (MS) analysis for the presence of propylhydroxybutyrate using a Kratos MS25 GC/MS. The following parameters were used:
30 source temperature, 210~C; voltage, 70eV; and accelerating voltage, 4 KeV.

W O 97/22711 ~CTiqUS96~201 Ig Catalytic activities.
Ketoacyl synthase (KS) activity was assessed radiochemically by the condensation-l4CO2 exchange reaction (Smith et al., PNAS USA Z~, 1184 (1976)).
Transferase (AT) activity was assayed, using malonyl-CoA
as donor and pantetheine as acceptor, by determining spectrophotometrically the free CoA released in a coupled ATP citrate-lyase-malate dehydrogenase reaction (see, Rangen et al., J. Biol.
Chem., 266 19180 (1991).
Ketoreductase (KR) was assayed spectrophotometrically at 340 nm: assay .~y~ellls contained Q.l M potassium phosphate buffer (pH 7), 0.15 mM NADPH, enzyme and either 10 mM trans-l-decalone or 0.1 mM acetoacetyl-CoA substrate.
Deh ydrase (DH) activ ity was assayed 15 spectrophotometrically at 270 nm using S-DL-~-hydroxybutyryl N-acetylcysteamine as substrate (Kumar et al., J. Biol. Chem., 245 4732 (1970)).
Enoyl reductase (ER) activity was assayed spectrophotometrically at 340 nm essentially as described by Strometal 20 (J. Biol. Chem. ~!, 8159 (1979)); the assay ~y~L~ contained 0.1 M
potassium phosphate buffer (pH 7), 0.15 mM NADPH, 0.375 mM
crotonoyl-CoA, 20 ~M CoA and enzyme.
Thioesterase (TE) activity was assessed radiochemically by extracting and assaying the ~l4C~palmitic acid formed from ~1-25 l4C]palmitoyl-CoA during a 3 minute incubation Smith, Meth.
Enzymol. ~, 181 (1981); the assay was in a final volume of 0.1 ml, 25 mM potassium phosphate buffer (pH 8), 20 ,uM [1-l4C]palmitoyl-CoA
(20 nCi) and enzyme.
Assay of overall fatty acid synthase activity was performed 30 spectrophotometrically as described previously by Smith et al. (Meth.

W O 97/22711 PCT~US96/20119 Enzymol., ~ 65 (1975)). All enzyme activities were assayed at 37~C
except the transferase, which was assayed at 20~C. Activity units indicate nmol of substrate consumed/minute. All assays were conducted, at a minimum, at two different protein concentrations 5 with the appropriate enzyme and substrate blanks included.

Example 1 Expression of A. eul;rophus PHA synthase using a baculovirus system.
Recent work has shown that PHA synthase from A.
eutrophZ~s can be overexpressed in E. coli, in the absence of 3-ketothiolase and acetoacetyl-CoA reductase (Gerngross et al. supra) and can be expressed in plants (See Poirier et al., Biotech ;~, 142 (1995) for a review). Isolation of the soluble form of PHA synthase provides 15 opportunities to examine the mechanistic details of the priming and initiation reactions. Because the baculovirus system has been successful for the expression of a number of prokaryotic genes as soluble proteins, and insect cells, unlike bacterial expression ~y~L~ms, carry out a wide array of posttranslational modifications, the 20 baculovirus expression ~ysl~ appeared ideal for the expression of large quantities of soluble PHA synthase, a protein that must be modified by phosphopantetheine in order to be catalytically active (Gerngross et al., supra).
Purification of PHA synthase. The purification 25 procedure employed for PHA synthase is a modification of Gerngross et al. (supr~) involving the elimination of the second liquid chromatographic step and inclusion of a protease-inhibitor cocktail in all buffers. All steps were carried out on ice or at 4~C except where noted. Frozen cells were thawed on ice in 10 ml of Buffer A (10 mM
30 KPi, pH 7.2, 0.5% glycerol, and 0.05% Hecameg) and then immediately homogenized prior to centrifugation and HA chromatography.

The results of these efforts are sunlm~ri7.ed in Table 1 and Figure 7. A prominent band at 64 kDa is visible. in total, soluble, and HA eluate protein samples fractionated by SDS/PAGE (lanes 4, 5, and 6 of Figure 7, respectively). The initial specific activity of the 5 isolated PHA synthase was 20-fold higher than previous attempts at expression and purification of this polypeptide. Approximately 1000 units of PHB synthase have been purified, based on calculations from the direct spectrophotometric assay detailed below, with an overall recovery of activity of 70%. The large proportion of synthase present 10 in the membrane fraction, and the fact that over 90% of the initial activity was found in the soluble fraction, suggests either that the synthase in the membrane fraction is in an inactive form or that the direct assay is not applicable to the initial, 12 U/mg, crude extract.

15 Table 1: Purification o~ PE~A Synthase protein specific sample total vol (mL) (mg) (mg/ml) activity recovery units total 1430 11.5 113 9.812.7 100 protein soluble 1340 10.5 47 4.528.6 93 protein pooled 1020 7.9 30 3.834.2 71 HA
fractions N-terminal sequencing of the 64 kDa protein confirmed its identity as PHA synthase (Figure 8). Two prominent N-termini, at an~ino acid residue 7 (alanine) and residue 10 (serine) were obtained in a 3:2 ratio. This heterogeneous N-terminus presumably is the result of aminopeptidase activity. Western analysis using a rabbit-W O 97122711 PCT~US96/20119 anti-PHA synthase antibody corroborated the results of the sequencing and indicated the presence of at least three bands that resulted from proteolysis of PHA synthase (Figure 7B, Lanes 4-6). The antibody was specific for PHA synthase since neither T. ni nor baculoviral proteins 5 showed reactivity (Figure 7B, Lanes 2 and 3). N-terminal protein sequencing (Figure 8) showed directly that the 44 kVa (band b) and 32 kDa (band d) proteins were derived from PHA synthase (fragrnents beginning at A181/N185 and at G387, respectively). The 35-40 kDa (band c) protein gave low sequencing yields and may contain a 10 blocked N-terminus. Inspection t)f Figure 7B suggests that most degradation occurs following cell disruption since the total protein sample for this gel (lane 4) was prepared by boiling intact cells directly in SDS sample buffer while the HA sample (lane 6) went through the purification procedure described above.
Assay of Synthase Activity. Due to the significant level of expression obtained using the baculovirus system, the synthase activity could be assayed spectrophotometrically by monitoring hydrolysis of the thioester bond at 232 nm, the wavelength at which there is a maximum decrease in absorbance upon hydrolysis The 20 difference between substrate (HBCoA) and product (CoA~ at this wavelength is shown in Figure 9. Absorbance of HBCoA and CoA at 232 nm occurs at a trough between two well separated peaks. Assays were carried out at pH 7.2 for comparative analysis with previous studies (Gerngross et al., supra). Substrate (R-(-)3-HBCoA) substrate for 25 these studies was prepared using the mixed anhydride method (Haywood et al., supra), and its concentration was determined by measuring A26o. The short pathlength cells (0.1 cm and 0.01 cm) allowed use of relatively high reaction concentrations while conserving substrate and enzyme. Assay results showed an initial lag 30 period of 60 seconds prior to the linear decrease in A 232, and velocities were determined from the slope of these linear regions of WO 97/22711 PCT~US96/207~9 the assay curves. The length of the lag period was variable and was inversely related to enzyme concentration. These data are consistent with those using PHA synthase purified from E. coli (Gerngross et al., su pra).
Figures 10 and 11 show the V versus S and 1/V versus 1 /S plots, respectively. The double reciprocal plot was concave upward which is similar to results obtained from studies of the granular PHA synthase from Zooglea ramigera (Fukui et al., ~rch.
~:icrobiol., 110 149 (1976)) and suggests a complex reaction mechanism. Examinations of velocity and specific activity as a function of enzyme concentration are shown in Figures 12 and 13.
These results cGnfirm that specific activity of the synthase depends upon enzyme concentration. The pH activity curve for A. eutrophus PHA synthase purified from T. ni cells is shown in Figure 14. The curve shows a broad activity maximum centered around pH 8.5. This result agrees well with prior work on the A. eutrophus PHB synthase although it is significantly different than results obtained for the PHB
synthase from Z. ramigera for which the optimum was determined to be pH 7Ø
The effect of varying enzyme concentration in the presenLce of a fixed amount of substrate revealed an intriguing trend (Figure 15). ~rom these data it appears that the extent of polymerization is dependent on the amount of enzyme included in the reaction mixture. This could be explained if there is a "terminal length" limitation of the polymer, which, once reached, can not be extended any further. If this is the case, it would also suggest that termination of the polymerization reaction, the release of the synthase from the polymer, and/or reinitiation of polymerization by the newly released synthase are relatively slow events since no evidence of these reactions are seen within the timecourse of these studies. The phenomenon observed in Figure 15 is not the result of W O 97/22711 PCT~US96/201~9 decay of the enzyme over the course of the assay since virtually identical results are obtained following a 10 minute preincubation of the synthase at 25~C.
It must also be noted that comparisons of the direct spectrophotomekic assays used here and the more common assay involving the use of Ellman's reagent, DTNB, (Ellman, supra) in the formation of thiolate of coenzyme-A showed that the values determined by the direct method were approximately 70% of the values determined using Ellman's reagent. This may be due to phase 10 separation occurring in the cuvettes as the relatively insoluble polymer is formed. In support of this notion, a faint haze or opalescence in the cuvette developed during the course of the reaction, particularly at higher substrate concentrations.
PHA synthase purified from insect cells appears to be 15 relatively stable. Examination of activity following storage, in liquid N 2 and at -20~C in the presence of 50% glycerol showed that approximately 50% of synthase activity remained after 7 weeks when stored in liquid N2 and approximately 75% of synthase activity remained after 7 weeks when stored at -20~C in the presence of 50%
20 glycerol.
The expression of PHA synthase from A. eutrophus in a baculovirus expression ~y~lem results in the synthase constituting approximately 50% of total protein 60 hours post-infection; however, approximately 50-75% of the synthase is observed in the membrane-25 associated fraction. This elevated level of expression allowed purification of the soluble PHA synthase using a single chromatographic step on HA. The purity of this preparation is estimated to be approximately 90% (intact PHA synthase and 3 proteolysis products).
The initial specific activity of 12 U/mg was approximately 20-fold higher than the most successful previous W O 97/22711 PCTfUS96~119 efforts at overexpression of A. eutrophus PHA synthase. The synthase reported here was isolated from a 250 ml culture with 70% recovery which represents an improvement of 500-fold (1000 U / 64 U x 8 L /
0.25 L) when compared to an 8 L E. coli culture with 40% recovery.
5 This high expression level should provide sufficient PHA synthase for extensive structural, functional, and mechanistic studies.
Furthermore, it is clear that the baculovirus expression ~y~lelll is an attractive option for isolation of other P~A synthases from various sources.
PHA synthase produced in the baculovirus system was of sufficient potency to allow direct spectrophotometric analysis of the hyd~olysis of the thioester bond of HBCoA at 232 nm. These assays revealed a lag period of approximately 60 seconds, the length of which was variable and inversely related to enzyme concentration. Such a 15 lag period presumably reflects a slow step in the reaction, perhaps correlating to dimerization of the enzyme, the priming, and/or initiation steps in formation of PHB. Size exclusion chromatographic examination of the PHB synthase native MW indicated two forms of the synthase. One form showed a MW of approximately 100-160 kDa 20 and the other showed a MW of approximately 50-80 kDa; these two forms likely represent the dimer and monomer of PHA synthase, respectively. SiI!nilar results have been reported previously in which two forms of approximately 60 and 130 kDa were observed.
Comparisons of the direct assay reported here and the indirect assay 25 using DTNB revealed that the former resulted in values that were 70% of the values determined by the DTNB indirect assay. Although the reason for this difference has not been examined in detail, it is probable that the apparent phase separation that occurred upon PHB
formation in the short pathlength cuvettes used, particularly with 30 high [~BCoA], results in this discrepancy.

W O 97/22711 PCT~US96/20119 Enzymatic analyses of the PHA synthase have found that the enzyme has a broad pH optimum centered at pH 8.5; however, the studies described herein have been performed at pH 7.2 to provide comparative values with the results of others. Moreover, the specific activity of this enzyme is dependent upon enzyme concentration which confirms and extends earlier results (Gerngross et al., supra).
In studies intended to examine the dependence of activity upon enzyme concentration, it became apparent that the extent of the polymerization reaction is dependent on the amount of 10 enzyme included in the reaction mixture. Specifically, decreasing the amount of enzyme leads not only to decreased velocity of reaction but also to a decreased extent of condensation ~Pigure 15). One possible explanation is that the enzyme is thermally labile; however, identical assays in which the enzyme is preincubated at 25~C for 10 minutes 15 prior to initiation of the reaction had similar results. Another possibility is that a terminal-length of the polymer is reached precluding further condensations until the particular synthase molecule is released from the terminal-length polymer.
This work clearly demonstrates the value of the 20 baculovirus expression ~ysl~ for the production of A. eutrophus PHA synthase and for the potential application to studies of other PHA synthases. Furthermore, the high level of expression obtained using the baculoviral system should allow convenient analysis for substrate-specificity and structure-function studies of PHA synthases 25 from relatively crude insect cell extracts.

Example 2 Co-expression of rat FAS dehydrase mutant cDNA and P~IB synthase gene in insect cells.
Expression of a rat FAS DH- cDNA in Sf9 cells has been reported previously (Rangan et al., J. Biol. Chem. 266, 19180 (1991);

WO 97/22711 PCTAUS96~20119 Joshi et al., Riochem. J., 296, 143 (1993)). Once activity of the phbC
gene product had been established in insect cells (see Example 1), baculovirus clones containing the rat FAS DH- cDNA and BacPAK6::phbC were employed in a double infection strategy to 5 determine if PHB would be produced in insect cells. It was not known if an intracellular pool of R(-)-3-hydroxybutyrate would be stable or available as a substrate for the PHB synthase. In order for the R-(-)-3-hydroxybutyrylCoA to be available as a substrate, the R-(-)-3-hydroxybutyry]CoA released from rat FAS DH- protein must be 10 trapped by the PHB synthase and incorporated into a polymer at a rate faster than ~-oxidation, which would regenerate acetylCoA. It was also not known if the stereochemical configuration of the 3-hydroxyl group, which must be in the R form, would be recognized as a substrate by PHB synthase. Fortunately, previous biochemical studies 15 on eukaryotic FASs indicated that the R form of 3-hydroxylbutyrylCoA would be generated (Wakil et al., J. Biol. Ch~
~, 687 (1962)).
SDS-PAGE of protein samples from a time course of uninfected, single-infected, and dual-infected Sf~l cells was performed 20 (Figure 16). From these data, it is clear that the rat FAS DH mutant and PHB synthase polypeptides are efficiently co-expressed in Sf21 cells. However, co-expression results in ~50% reduced levels of both polypeptides compared to Sf 21 cells that are producing the individual proteins. Western analysis using anti-rat FAS (Rangan et al., supra) 25 and anti-PHA synthase antibodies confirmed simultaneous ~ production of the corresponding proteins.
To provide further evidence that PHB was being synthesized in insect cells, T. ni cells which had been infected with a baculovirus vector encoding rat FAS DH~ and/or a baculovirus vector 3~ encoding PHA synthase were analyzed for the presence of granules.

CA 02240360 l998-06-ll W O 97/22711 PCT~US96/20119 Infected cells were fixed in paraformaldehyde and incubated with anti-PHA synthase antibodies (Williams et al., Protein Exp. Purif. Z, 203 (19~6)). Granules were observed only in doubly infected cells (Williams et al., App. Environ. Micro., ~2, 2540 (1996)).
(~haracterization of PHB production in insect cells. In order to determine if de novo synthesis of PHB was occurring in S~21 cells that co-express the rat FAS D~I mutant and PHB synthase, fractions of these samples were extracted, the extract subjected to propanolysis, and analyzed for the presence of propylhydroxybutyrate 10 by gas chromatography (Figure 17). A unique peak with a retention tin e that coincided with a propylhydroxybutyrate standard was detected only in the double infection samples at 48 and 72 hours, in contrast to the individually expressed gene products and uninfected controls, which were negative. These samples were analyzed further 15 by GC/MS to confirm the identity of the product. Figure 18 shows mass spectroscopy data corresponding to the material obtained from peak 10.1 in the gas chromatograph compared to an propylhydroxybutyrate standard. The results show that PHB synthesis is occurring only in Sf21 cells co-expressing the rat FAS DH mutant 20 cDNA and the phbC gene from A. eutrophzls. Integration of the peak in the gas chromatograph corresponding to propylhydroxybutyrate revealed that approximately 1 mg of PHB was isolated from 1 liter culture of Sf~1 cells (approximately 600 mg dry cell weight of Sf~1 cells). Thus, the ratFAS206 protein effectively replaces the ,B-25 ketothiolase and acetoacetyl-CoA reductase functions, resulting in the production of PHB by a novel pathway.
The approach described here provides a new strategy to combine metabolic pathways that are normally engaged in primary anabolic functions for production of polyesters. The premature 30 termination of the normal fatty acid biosynthetic pathway to provide suitably modified acylCoA monomers for use in PHA synthesis can be applied to both prokaryotic and eukaryotic expression since the formation of polymer will not be dependent on specialized feedstocks.
Thus, once a recombinant PHA monomer synthase is introduced into 5 a prokaryotic or eukaryotic system, and co-expressed with the appropriate PEIA synthase, novel biopolymer formation can occur.

Example 3 Cloning and Sequencing of the vep ORFI PKS Gene Cluster The entire PKS cluster from Strepfomyces venezuelae was cloned using a heterologous hybridization strategy. A 1.2 kb DNA
fragment that hybridized strongly to a DNA encoding an eryA PKS ,~-ketoacyl synthase domain was cloned and used to generate a pl~.~mi~l for gene disrupltion. This method generated a mutant strain blocked in the synthesis of the antibiotic. A S. venezue~ae genomic DNA
library was generated. and used to clone a cosmid containing the complete methymycin aglycone PKS DNA. Fine-mapping analysis was performed to identify the order and sequence of catalytic domains along the multifunctional PKS (Figure 19). DNA sequence analysis of the v e p ORFI showed that the order of catalytic domains is KSQ/AT/ACP/KS/AT/KR/ACP/KS/AT/DH/KR/ACP. The complete DNA sequence, and corresponding amino acid sequence, of the vep O~FI is shown in Pigure 23 (SEQ ID NO:1 and SEQ ID NO:2, respcctively).
The sequence data indicated that the PKS gene cluster encodes a polyene of twelve carbons. The vep gene cluster contains 5 polyketide synthase modules, with a loading module at its 5' end and an ending domain at its 3' end. Each of the sequenced modules includes a keto-ACP (KS), an acyltransferase (AT), a dehydratase (DH), W O 97J22711 PCT~US96/20119 a keto-reductase (KR), and an acyl carrier protein domain. The six acyltrans~erase domains in the cluster are responsible for the incorporation of six acetyl-CoA moieties into the product. I'he loading module contains a KSQ, an AT and an ACP domain. KSQ

5 refers to a domain that is homologous to a KS domain except that the active site cysteine (C) is replaced by glutamine (Q). There is no counterpart to the KSQ domain in the PKS dusters which have been previously characterized.
The ending domain (ED) is an enzyme which is 10 responsible for the attachment of the nascent polyketide chain onto another molecule. The amino acid sequence of ED resembles an enzyme, HetM, which is involved in Anabaen~ heterocyst formation.
The homology between vep and HetM suggests that the polypeptide encoded by the vep gene cluster may synthesize a polyene-containing 15 composition which is present in the spore coat or cell wall of its natural host, S. venez~elae.

Example 4 To provide a recombinant monomer synthase that 20 generates a saturated ~-hydroxyhexanoylCoA or unsaturated ,~-hydroxyhexanoylCoA monomer, the linear correspondence between the genetic organization of the Type I macrolide PKS and the catalytic domain organization in the multifunctional proteins is assessed (Donadio et al., supra, 1991; Katz et al., Ann. Rev. Microbiol., ~, 875 25 (1993)). First, a DNA encoding a TE is added to the 3' end of an ORFI
of a Type I PKS, preferably the met ORF I (Figure 6) as recently described by C~ortes et al. (Science. 268, 1487 (1995) in the erythromycin system. To ensure that the DNA encoding the TE is completely active, DNA encoding a linker region separating a normal ACP-TE
30 region in a PKS, for example the one found in met PKS ORF5 (~igure W O 97/22711 PCT~US96~01I9 5), will be incorporated into the DNA. The resulting vector can be introduced into a host cell and the TE activity, rate of release of the CoA product, and identity of the fatty acid chain determined.
The acyl chain that is most likely to be released is the CoA
5 ester, specifically the 3-hydroxy-4-methyl heptenoylCoA ester, since the fully elongated chain is presumably released in this form prior to macrolide cyclization. If the CoA form of the acyl chain is not observed, then a gene encoding a CoA ligase will be cloned and co-expressed in the host cell to catalyze formation of the desired 10 intermediate.
There is clear precedent for release of the predicted premature termination products from mutant strains of macrolide-producing Streptomyces that produce intermediates in macrolide synthesis (Huber et al., Antimicrob. A~ents Chemother. ~, 1535 15 (1990); Kinoshita et al., J. Chem. Soc. Chem. Comm., 14, 943 (1988)).
The structure of these intermediates is consistent with the linear organization of functional domains in macrolide PKSs, particularly those related to eryA, tyl, and mef. Other known PKS gene clusters include, but are not limited to, the gene cluster encoding 6-20 methysalicylic acid synthase (Beck et al., Eur. J. Biochem., ~, 487(1990)), soraphen A (Schupp et al., J. Bacteriol. 177, 3673 t1995), and sterigmatocystin (Yu et al., J. Bacteriol. 177 4792 (1995)).
Once the release of the 3-hydroxy-4-methyl heptenoylCoA
ester is established, DNA encoding the extender unit AT in m e t 25 module 1 is replaced to change the specificity from methylmalonylCoA to malonylCoA (Figures 4-6). This change eliminates methyl group branching in the ,B-hydroxy acyl chain.
While comparison of known AT amino acid sequences shows high overall amino acid sequence conservation, distinct regions are readily 30 apparent where significant deletions or insertions have occurred. For CA 02240360 1998-06-ll W O 97/22711 PCT~US96/20119 example, comparison of malonyl and methylmalonyl amino acid sequences reveals a 37 amino acid deletion in the central region of the malonyltransferase. Thus, to change the specificity of the methylmalonyl transferase to malonyl transferase, the met ORFI
5 DNA encoding the 37 amino acid sequence of MMT will be deleted, and the resulting gene will be tested in a host cell for production of the desmethyl species, 3-hydroxyheptenoylCoA. Alternatively, the DNA encoding the entire MMT can be replaced with a DNA encoding an intact MT to affect the desired chain construction.
After replacing MMT with MT, DNA encoding DH/ER
will be introduced into DNA encoding met ORFI module 1. This modification results in a multifunctional protein that generates a methylene group at C-3 of the acyl chain (Figure 6). The DNA
encoding DH/ER will be PCR amplified from the available eryA or tyl 15 PKS sequences, including the DNA encoding the required linker regions, employing a primer pair to conserved sequences 5' and 3' of the DNA encoding DH/ER. The PCR fragment will then be cloned into the mef ORFI. The result is a DNA encoding a multifunctional protein (MT# DH/ER~TE*). This protein possesses the full 20 complement of keto group processing steps and results in the production of heptenoylCoA.
The DNA encoding dehydrase in met module 2 is then inactivated, using site-directed mutagenesis in a scheme similar to that used to generate the rat FAS DH- described above (Joshi et al., L
25 Biol. Chem., 268, 22508 (1993)). This preserves the required (R)-3-hydroxy group which serves as the substrate for PHA synthases and results in a (R~-3-hydroxyheptanoylCoA species.
The final domain replacement will involve the DNA
encoding the starter unit acyltransferase in met module 1 (Figure 5), 30 to change the specificity from propionyl CoA to acetyl CoA. This shortens the (R)-3-hydroxy acyl chain from heptanoyl to hexanoyl.

The DNA encoding the catalytic domain will need to be generated based on a FAS or 6-methylsalicylic acid synthase model (Beck et al., Fllr. J. Biochem. ~2, 487 ~1990)) or by using site-directed mutagenesis to alter the specificity of the resident met PKS propionyltransferase 5 sequence. Limiting the initiator species to acetylCoA can result in the use of this starter unit by the monomer synthase. Previous work with macrolide synthases have shown that some are able to accept a wide range of starter unit carboxylic acids. This is particularly well documented for avermectin synthase, where over 60 new compounds 10 have been produced by altering the starter unit substrate in precursor feeding studies (Dutton et al., J. Antibiotics, 44, 357 (1991)).

l~xample 5 To provide a recombinant monomer synthase that 15 synthesizes 3-hydroxyl-4-hexenoic acid, a precursor for polyhydoxyhexenoate, the DNA segment encoding the loading and the first module of the vep gene cluster was linked to the DNA
segment encoding module 7 of the tyZ gene cluster so as to yield a recombinant DNA molecule encoding a fusion polypeptide which has 20 no amino acid di~fences relative to the corresponding amino acid sequence of the parent modules. The fusion polypeptide catalyzes the synthesis of 3-hydroxyl-4-hexenoic acid. The recombinant DNA
molecule was introduced into SCP2, a Streptomyces vector, under the control of the a c t promoter (pDHS502, Figure 20) . A
25 polyhydroxyalkanoate polymerase gene, phaC1 from Pseudomonas oleavorans, was then introduced downstream of the recombinant PKS cluster (pDHS505; Figures 22 and 23). The DNA segment encoding the polyhydroxyalkanoate polymerase is linked to the DNA
segment encoding the recombinant PKS synthase so as to yield a 30 fusion polypeptide which synthesizes polyhydroxyhexenoate in Strep~omyces. Polyhydroxyhexenoate, a biodegradable thermoplastic, W O 97/22711 PCT~US96/20119 is not naturally synthesized in Streptomyces. or as a major product in any other organism. Moreover, the unsaturated double bond in the side chain of polyhydroxyhexenoate may result in a polymer which has superior physical properties as a biodegradable thermoplastic over 5 the known polyhydroxyalkanoates.
The complete disclosure of all patents, patent documents and publications cited herein are incorporated herein by reference as if individually incorporated. The foregoing detailed description and examples have been given for clarity of understanding only. No 10 unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims (58)

WHAT IS CLAIMED IS:

1. A baculovirus expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in an insect cell.

2. The expression cassette of claim 1 wherein the source of the nucleic acid molecule is a bacterium.

3. The expression cassette of claim 2 wherein the bacterium is Alcaligenes eutrophus.

4. An expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell, wherein the nucleic acid molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea.

5. The expression cassette of claim 4 wherein the source of at least one DNA segment is bacterial DNA.

6. A method of providing a polyhydroxyalkanoate synthase, comprising:
(a) introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase, operably linked to a promoter functional in a eukaryotic cell into the eukaryotic cell, wherein the eukaryotic cell is not of plant origin; and (b) expressing the DNA molecule encoding the polyhydroxyalkanoate synthase in the eukaryotic cell.

7. The method of claim 6 wherein the polyhydroxyalkanoate synthase is polyhydroxybutyrate synthase.

8. The method of claim 6 wherein the polyhydroxyalkanoate synthase is derived from a bacterium.

9. The method of claim 8 wherein the bacterium is Alcaligenes eutrophus.

10. The method of claim 6 wherein the eukaryotic cell is of insect origin.

11. The method of claim 10 wherein the expression cassette is a baculovirus expression cassette.

12. The method of claim 6 further comprising isolating polyhydroxyalkanoate synthase from the eukaryotic cell.

13. A method of providing a polyhydroxyalkanoate polymer, comprising:
(a) introducing into a eukaryotic cell (i) a first expression cassette comprising a DNA segment encoding a fatty acid synthase in which the dehydrase activity is inactivated, wherein said segment is operably linked to a promoter functional in the eukaryotic cell, and (ii) a second expression cassette comprising a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the eukaryotic cell; and (b) expressing the DNA segments so as to yield a polyhydroxyalkanoate polymer in the eukaryotic cell.

14. The method of claim 13 wherein the eukaryotic cell is of insect origin.

15. The method of claim 13 wherein the dehydrase activity is inactivated by mutating the catalytic site.

16. The method of claim 13 wherein the fatty acid synthase is a rat fatty acid synthase.

17. The method of claim 13 wherein the polyhydroxyalkanoate synthase is a polyhydroxybutyrate synthase.

18. The method of claim 13 wherein the fatty acid synthase produces a premature termination product.

19. The method of claim 13 wherein the fatty acid synthase catalyzes the synthesis of D(-)-3-hydroxybutyrate in the eukaryotic cell.

20. The method of claim 13 wherein the polyhydroxyalkanoate polymer is polyhydroxybutyrate.

21. The method of claim 13 wherein the first and second expression cassettes are on different DNA molecules.

22. An isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea.

23. The isolated DNA molecule of claim 22 wherein the first DNA segment is derived from the vep gene cluster of Streptomyces venezuelae.

24. The isolated DNA molecule of claim 22 wherein the second DNA
segment is derived from the tyl gene cluster of Streptomyces.

25. The isolated DNA molecule of claim 22 wherein the second DNA
segment comprises a DNA encoding a thioesterase which is located at the 3' end of the second DNA segment.

26. The isolated DNA molecule of claim 25 wherein the second DNA
segment comprises a DNA encoding an acyl carrier protein which is located 5' to the DNA encoding the thioesterase.

27. The isolated DNA molecule of claim 26 wherein the second DNA
segment comprises a DNA encoding a linker region, wherein the DNA
encoding the linker region is located between the DNA encoding the acyl carrier protein and the DNA encoding the thioesterase.

28. The isolated DNA molecule of claim 22 wherein the first DNA segment comprises DNA encoding two acyl transferases, wherein the DNA
encoding the first acyl transferase is 5' to the DNA encoding the second acyl transferase.

29. The isolated DNA molecule of claim 28 wherein the second acyl transferase adds acyl groups to malonyl CoA.

30. The isolated DNA molecule of claim 22 wherein the first DNA segment comprises a DNA encoding a dehydrase.

31. The isolated DNA molecule of claim 22 wherein the first DNA segment comprises a DNA encoding a dehydrase and an enoyl reductase.

32. The isolated DNA molecule of claim 22 wherein the second DNA
segment comprises a DNA encoding an inactive dehydrase.

33. The isolated DNA molecule of claim 22 wherein the first DNA segment comprises a DNA encoding an acyl transferase.

34. The isolated DNA molecule of claim 33 wherein the acyl transferase domain binds an acyl CoA substrate.

35. The isolated DNA molecule of claim 22 wherein the first DNA segment encodes the first module from the vep gene cluster and the second DNA
segment encodes module 7 from the tyl gene cluster.

36. A method of providing a polyhydroxyalkanoate monomer, comprising:
(a) introducing into a host cell a DNA molecule comprising a DNA
segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell, wherein the recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module; and (b) expressing the DNA encoding the recombinant polyhydroxyalkanoate monomer synthase in the host cell so as to generate a polyhydroxyalkanoate monomer.

37. A method of providing a polyhydroxyalkanoate polymer, comprising:
(a) introducing into a host cell I) a first DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell, and ii) a second DNA molecule comprising a DNA
segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell, wherein the recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module; and (b) expressing the DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase in the host cell so as to generate a polyhydroxyalkanoate polymer.

38. The method of claim 36 or 37 wherein the first DNA segment encodes the first module from the vep gene cluster and the second DNA segment encodes module 7 from the zyl P gene cluster.

39. An isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module of polyketide synthase.

40. The isolated DNA molecule of claim 39 wherein the second DNA
segment encodes a .beta.-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.

41. The isolated DNA molecule of claim 39 wherein the second DNA
segment is 3' to the DNA encoding the fatty acid synthase.

42. The isolated DNA molecule of claim 39 wherein the second DNA
segment is separated from the first DNA segment by a DNA encoding a linker region.

43. The isolated DNA molecule of claim 40 wherein the DNA encoding the linker region is selected from the group consisting of tyl ORF1 ACP1-KS2, tyl ORF1 ACP2-KS3, tyl ORF3 ACP5-KS6 eryA ORF1 ACP1-KS1, eryA ORF1 ACP2-KS2, eryA ORF2 ACP3-KS4, and eryA ORF2 ACP5-KS6.

44. A method of providing a polyhydroxyalkanoate monomer, comprising:

(a) introducing into a host cell a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA
segment encoding a polyketide synthase, wherein the first DNA
segment is 5' to the second DNA segment, wherein the first DNA
segment is operably linked to a promoter functional in the host cell, and wherein the first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein; and (b) expressing the DNA molecule in the host cell so as to generate a polyhydroxyalkanoate monomer.

45. The method of claim 44 wherein the host cell is selected from the group consisting of insect cells, Streptomyces cells and Pseudomonas cells.

46. The method of claim 44 wherein the DNA encoding the fatty acid synthase is eukaryotic in origin.

47. The method of claim 44 wherein the DNA encoding the fatty acid synthase is prokaryotic in origin.

48. The method of claim 44 wherein the DNA encoding the polyketide synthase module is derived from DNA encoding the tyl module F.

49. An expression cassette comprising a DNA molecule comprising a DNA
segment encoding a fatty acid synthase and a polyhydroxyalkanoate synthase.

50. A method of providing a polyhydroxyalkanoate monomer synthase, comprising:
(a) introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell, wherein the DNA comprises a first DNA segment encoding a first module and a second DNA
segment encoding a second module wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase; and (b) expressing the DNA molecule in the host cell.

51. An isolated and purified DNA molecule comprising a DNA segment encoding a Streptomyces venezuelae Type 1 polyketide synthase.

52. The isolated DNA molecule of claim 51 wherein the DNA segment comprises SEQ ID NO:1.

53. The isolated DNA molecule of claim 51 wherein the DNA segment encodes a polypeptide having an amino acid sequence comprising SEQ
ID NO:2.

54. The expression cassette of claim 4 wherein the first DNA segment encodes the first module from the vep gene cluster and the second DNA
segment encodes module 7 from the tyl P gene cluster.

55. The expression cassette of claim 4 further comprising a third DNA
segment encoding a polyhydroxyalkanoate synthase.

56. The method of claim 36 wherein the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase.

57. The isolated DNA molecule of claim 22 or 35 further comprising a DNA
segment encoding a polyhydroxyalkanoate synthase.

58. The method of claim 50 wherein the expression cassette further comprises a second DNA molecule encoding a polyhydroxyalkanoate synthase.