Identification and Analysis of the Polyhydroxyalkanoate-Specific β-Ketothiolase and Acetoacetyl Coenzyme A Reductase Genes in the Cyanobacterium Synechocystis sp. Strain PCC6803

Synechocystis sp. strain PCC6803 possesses a polyhydroxyalkanoate (PHA)-specific β-ketothiolase encoded by phaA_Syn and an acetoacetyl-coenzyme A (CoA) reductase encoded by phaB_Syn. A similarity search of the entire Synechocystis genome sequence identified a cluster of two putative open reading frames (ORFs) for these genes, slr1993 and slr1994. Sequence analysis showed that the ORFs encode proteins having 409 and 240 amino acids, respectively. The two ORFs are colinear and most probably coexpressed, as revealed by sequence analysis of the promoter regions. Heterologous transformation of Escherichia coli with the two genes and the PHA synthase of Synechocystis resulted in accumulation of PHAs that accounted for up to 12.3% of the cell dry weight under high-glucose growth conditions. Targeted disruption of the above gene cluster in Synechocystis eliminated the accumulation of PHAs. ORFs slr1993 and slr1994 thus encode the PHA-specific β-ketothiolase and acetoacetyl-CoA reductase of Synechocystis and, together with the recently characterized PHA synthase genes in this organism (S. Hein, H. Tran, and A. Steinbüchel, Arch. Microbiol. 170:162–170, 1998), form the first complete PHA biosynthesis pathway known in cyanobacteria. Sequence alignment of all known short-chain-length PHA-specific acetoacetyl-CoA reductases also suggests an extended signature sequence, VTGXXXGIG, for this group of proteins. Phylogenetic analysis further places the origin of phaA_Syn and phaB_Syn in the γ subdivision of the division Proteobacteria.     

Analysis of β-ketothiolase and acetoacetyl-CoA reductase genes of a methylotrophic bacterium, Paracoccus denitrifleans, and their expression in Escherichia coli

Abstract The β-ketothiolase gene ( phaA ) and acetoacetyl-CoA reductase gene ( phaB ) were isolated from Paracoccus denitrificans . Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40744 Da and of 242 amino acids with a molecular mass of 25614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4–74.0% for the phaA gene product and 47.6–80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli . In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA - phaB , in P. denitrificans . NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.     

丛毛单胞菌CNB-1合成PHA及相关基因的研究

分析了丛毛单胞菌(Comamonas sp.)CNB-1菌株在不同条件下合成聚羟基烷酸(polyhydroxyalkanoic acids,PHAs)的组分和含量,同时克隆了与PHA合成相关的基因。结果表明,该菌可以多种短链有机酸及醇类为碳源合成PHA多聚物或共聚物,以戊酸和1,4-丁二醇为底物时,可达菌体干重的57%;同时发现小分子醇类的存在能显著促进PHA的合成,推测与醇类氧化过程中提供了更多的还原力有关。为了克隆相关基因,利用已知phaC的保守区简并引物筛选基因组文库,将得到的阳性克隆质粒测序,发现phaC、phaA、phaB组成一个基因簇phaC-A-B。将phaC、phaA、phaB连接到pET载体在E.coli中共表达,重组E.coli菌株能合成PHA;将这3个基因单独连接到pET载体,在E.coli中表达后检测到相应酶活,分别约为原始菌株的4.1、71和2882倍。     

Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C(1) and C(2) compounds in the methylotroph Methylobacterium extorquens AM1

Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.     

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.     

Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109

In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738bp) and PHA synthase, class I (phaC(Co) gene, 1694bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33wt.% and 17wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.     

突变工程菌pCBH4高产PHAs分子机制的初步研究(英文)

聚羟基烷酸酯(PHAs)是一类以胞内颗粒形式存在的能被完全生物降解的贮能物质.本研究利用生物信息学的方法初步分析了离子束诱变选育出的突变工程菌pCBH4高产PHAs分子机制.为了分析高产PHAs特性,实验首先提取pCBH4突变菌株含有合成PHAs基因的质粒并转化受体菌DH5α,然后根据原始序列设计引物,从突变株pCBH4中扩增出phaA,phaB和phaC基因并对其进行了序列分析.结果表明:转化菌表现出高产PHAs特性;phaA基因在序列末端有7处碱基发生改变,这一变化导致5个氨基酸发生了改变;phaC基因也发生了类似的变化.从这些结果可以推断pCBH4突变株高产PHAs不是由于工程菌基因组遗传变异所致,可能是phaA和phaC基因突变的结果.     

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.     

Insight in to the phylogeny of polyhydroxyalkanoate biosynthesis: horizontal gene transfer

Polyhydroxyalkanoates (PHAs) are gaining more and more importance the world over due to their structural diversity and close analogy to plastics. Their biodegradability makes them extremely desirable substitutes for synthetic plastics. PHAs are produced in organisms under certain stress conditions. Here, we investigated 253 sequenced (completely and unfinished) genomes for the diversity and phylogenetics of the PHA biosynthesis. Discrepancies in the phylogenetic trees for phaA, phaB and phaC genes of the PHA biosynthesis have led to the suggestion that horizontal gene transfer (HGT) may be a major contributor for its evolution. Twenty four organisms belonging to diverse taxa were found to be involved in HGT. Among these, Bacillus cereus ATCC 14579 and Xanthomonas axonopodis pv. citri str. 306 seem to have acquired all the three genes through HGT events and have not been characterized so far as PHA producers. This study also revealed certain potential organisms such as Streptomyces coelicolor A3(2), Staphylococcus epidermidis ATCC 12228, Brucella suis 1330, Burkholderia sp., DSMZ 9242 and Leptospira interrogans serovar lai str. 56601, which can be transformed into novel PHA producers through recombinant DNA technology.     

Dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in Ralstonia eutropha

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation. The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.     

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by a wide range of bacteria as carbon and energy reserves. PHAs are typically stored when organisms are in an environment in which carbon is plentiful but the lack of another nutrient limits normal cell growth. It has been found that in environments with fluctuating carbon levels, PHA producers have crucial advantages over rival species (14). In addition to their importance in the microbial world, these polymers have been studied for their potential uses in biodegradable consumer goods (12) and medical products (22) and as chemical precursors (4). Although many PHA monomers have been discovered, the most common are 3-hydroxyalkanoates (32). Common PHAs are typically characterized by their constituent monomers as short-chain-length polymers (SCL-PHA; C₄ and C₅ monomers) or medium-chain-length polymers (MCL-PHA; C₆ and longer monomers).The model organism used to study PHA biosynthesis is the Gram-negative bacterium Ralstonia eutropha. This organism accumulates a high percentage of its cell dry weight (CDW) as SCL-PHA under nutrient limitation. When grown on sugars or plant oils, R. eutropha makes poly(3-hydroxybutyrate) (PHB) almost exclusively, although the addition of precursors such as propionate to the growth medium can lead to incorporation of 3-hydroxyvalerate into the polymer chain as well (2). An operon of biosynthetic genes from R. eutropha encoding enzymes sufficient for synthesis of PHB from acetyl coenzyme A (acetyl-CoA), which consisted of phaC-phaA-phaB, was discovered in the late 1980s (25, 26, 36). In this pathway, two molecules of acetyl-CoA are condensed by a β-ketothiolase (PhaA) and the resulting acetoacetyl-CoA is reduced by a reductase (PhaB) to form (R)-3-hydroxybutyryl-CoA (HB-CoA), which is the substrate for the PHA synthase (PhaC). Sequencing and analysis of the R. eutropha genome revealed the existence of putative isologs for each of the PHA synthetic genes (29). While the existence of alternate β-ketothiolases was already known (39), most of the potential isologs identified had never been characterized.Our group wanted to better understand how acetoacetyl-CoA reduction occurs in R. eutropha. In addition to the earlier-identified phaB gene, now referred to as phaB1 (GeneID, 4249784), the genes phaB2 (GeneID, 4249785) and phaB3 (GeneID, 4250155) were discovered on R. eutropha chromosome 1. Fifteen other potential isologs were also found to encode amino acid sequences that could potentially indicate acetoacetyl-CoA reductase activity (29). The roles of the newly discovered genes in PHB biosynthesis were unclear, especially given the results of an earlier biochemical study that suggested there was a single NADPH-dependent acetoacetyl-CoA reductase in R. eutropha (10). In order to determine the roles of the reductase genes in R. eutropha, we deleted phaB1, phaB2, and phaB3 from the genome both individually and in combination. In addition to characterizing these newly discovered genes, we also hoped to eliminate or diminish formation of HB-CoA by stopping the reduction reaction. Efforts to purify the PHA synthase from R. eutropha have been complicated by the high levels of PHB made by this organism (7). Studying formation and growth of PHB granules is difficult because PHB accumulates at a high rate, causing individual granules to coalesce and become indistinct (44). We therefore believed that an R. eutropha strain with decreased HB-CoA synthesis would be a useful experimental tool and could also serve as a platform for engineering new PHA synthesis pathways into R. eutropha.     

嗜水气单胞菌合成含3-羟基戊酸单体的聚羟基脂肪酸共聚酯的研究

分别利用葡萄糖或葡萄糖酸钠与十一碳酸、月桂酸与十一碳酸为混合碳源进行嗜水气单孢菌 (Aeromonashydrophila)菌株 4AK4的摇瓶培养 ,实现了含有 3 羟基戊酸 (3HV)单体的聚羟基脂肪酸酯的微生物合成。当使用葡萄糖或葡萄糖酸钠与十一碳酸为混合碳源时 ,野生型A .hydrophila 4AK4及含有 3 羟基丁酸辅酶A合成基因phaA和phaB的重组A .hydrophila 4AK4 (pTG01)能够合成-3-羟基丁酸(3HB)与-3HV的共聚物 ,且葡萄糖或葡萄糖酸钠与十一碳酸比例为 1∶1时最利于细胞生长和PHA的积累。当使用月桂酸和十一碳酸为混合碳源时 ,A .hydrophila4AK4能够合成-3HB、3HV与 β-羟基己酸 (3HHx)的共聚物 ,且随着混合碳源中十一碳酸的含量增加 ,A .hydrophila4AK4合成的PHA中-3HV的比例增加 ,而-3HB和-3HHx的比例降低.     

Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase

The poly-beta-hydroxybutyrate biosynthetic thiolase gene from Zoogloea ramigera was used as a hybridization probe to screen restriction digests of Alcaligenes eutrophus H16 DNA. Specific hybridization signals were obtained and two fragments (a 2.3-kilobase PstI fragment and a 15-kilobase EcoRI fragment) cloned in the Escherichia coli vector pUC8 (plasmids pAeT3/pAeT10 and pAeT29, respectively). Biochemical analysis of lysates of E. coli cells containing each plasmid identified significant levels of beta-ketothiolase and acetoacetyl-CoA reductase enzyme activities in lysates of E. coli cells containing plasmids pAeT10 or pAeT29. Nucleotide sequence analysis of the pAeT10 insert identified two open reading frames which encode polypeptides of Mr = 40,500 and Mr = 26,300 corresponding to the structural genes for beta-ketothiolase (phbA) and acetoacetyl-CoA reductase (phbB), respectively. Amino acid sequence homologies between the two bacterial and two mammalian thiolases are discussed with respect to the chain length specificity exhibited by the different thiolase enzymes.     

Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli

Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.     

Metabolic engineering of Alcaligenes eutrophus through the transformation of cloned phbCAB genes for the investigation of the regulatory mechanism of polyhydroxyalkanoate biosynthesis

The regulatory mechanisms of the biosynthesis of in vivo poly-beta-hydroxybutyrate [PHB] and poly(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] of Alcaligenes eutrophus were investigated by using various transformants with enzyme activities that were modified through the transformation of cloned phbCAB genes. The biosynthesis rates of PHB and P(3HB-3HV) were controlled by beta-ketothiolase and acetoacetyl-CoA reductase, and especially by beta-ketothiolase condensing acetyl-CoA or propionyl-CoA. The contents of PHB and P(3HB-3HV) were controlled by PHB synthase, polymerizing 3-hydroxybutyrate to PHB or 3-hydroxybutyrate and 3-hydroxyvalerate to P(3HB-3HV). The molar fraction of 3-hydroxyvalerate in P(3HB-3HV) was also closely connected with PHB synthase. This may be due to the accelerated polymerization between 3-HB from glycolysis pathway and 3-HV converted from propionate supplied as precursor. Enforced beta-ketothiolase and acetoacetyl-CoA reductase to PHB synthase tended to enlarge the size of the PHB and P(3HB-3HV) granules, however, higher activity ratio of PHB synthase to beta-ketothiolase and acetoacetyl-CoA reductase than parent strain tended to induce the number of granules.     

Effective enhancement of short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production by coexpression of genetically engineered 3-ketoacyl-acyl-carrier-protein synthase III (fabH) and polyhydroxyalkanoate synthesis genes

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that have a wide variety of physical properties dependent on the lengths of the pendant groups of the monomer units in the polymer. PHAs composed of mostly short-chain-length (SCL) monomers are often stiff and brittle, whereas PHAs composed of mostly medium-chain-length (MCL) monomers are elastomeric in nature. SCL-MCL PHA copolymers can have properties between the two states, dependent on the ratio of SCL and MCL monomers in the copolymer. It is desirable to elucidate new and low cost ways to produce PHA composed of mostly SCL monomer units with a small mol % of MCL monomers from renewable resources, since this type of SCL-MCL PHA copolymer has superior qualities compared to SCL homopolymer. To address this issue, we have created strains of recombinant E. coli capable of producing beta-ketothiolase (PhbA) and acetoacetyl-CoA synthase (PhbB) from Ralstonia eutropha, genetically engineered 3-ketoacyl-ACP synthase III (FabH) from Escherichia coli, and genetically engineered PHA synthases (PhaC) from Pseudomonas sp. 61-3 to enhance the production of SCL-MCL PHA copolymers from glucose. The cumulative effect of having two monomer-supplying pathways and genetically engineered PHA synthases resulted in higher accumulated amounts of SCL-MCL PHA copolymer from glucose. Polymers were isolated from two recombinant E. coli strains, the first harboring the phbAB, fabH(F87T), and phaC1(SCQM) genes and the second harboring the phbAB, fabH(F87W), and phaC1(SCQM) genes. The thermal and physical properties of the isolated polymers were characterized. It was found that even a very low mol % of MCL monomer in a SCL-MCL PHA copolymer had dramatic effects on the thermal properties of the copolymers.     

Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.