Human deoxycytidine kinase as a conditional mutator in Escherichia coli

The chemical diversification of DNA precursors was undertaken in Escherichia coli by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar. Arabinocytidine and dideoxycytidine thus became highly toxic to E. coli in the sub-millimolar range. Deoxynucleosides bearing isoadenine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ). These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo.     

Expanding the landscape of recombinant protein production in Escherichia coli

Over the years, several vectors and host strains have been constructed to improve the overexpression of recombinant proteins in Escherichia coli. More recently, attention has focused on the co-expression of genes in E. coli, either by means of a single vector or by cotransformation with multiple compatible plasmids. Co-expression was initially designed to generate protein complexes in vivo, and later served to extend the use of E. coli as a platform for the production of heterologous proteins. This review shows how the co-expression of genes in E. coli is challenging the production of protein complexes and proteins bearing post-translational modifications or unnatural amino acids. In addition, the importance of co-expression to achieve efficient secretion of recombinant proteins in E. coli is discussed, with recent insights into the use of co-expression to overproduce membrane proteins.     

Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10^?8/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10^?8/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.     

Enhancing survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> by increasing the periplasmic expression of Cu,Zn superoxide dismutase from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The gene for the Cu,Zn superoxide dismutase (Cu,ZnSOD) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli LMG194. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the E. coli periplasmic expression vector pBAD/gIIIA, yielding pBAD-1. E. coli was transformed using the constructed plasmid pBAD-1 and induced by adding 0.02% l-arabinose to express Cu,ZnSOD protein. The results indicated that Cu,ZnSOD enzyme activity in the periplasmic space was about fivefold to sixfold higher in the recombinant E. coli strains bearing the sod gene than in the control strains. The yields of Cu,ZnSOD were about threefold higher at 48 h than at 24 h in the recombinant E. coli cells. Significantly higher survival of strains was obtained in cells bearing the sod gene than in the control cells when the cells were treated by heat shock and superoxide-generating agents, such as paraquat and menadione.     

Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.     

Metabolic engineering of Escherichia coli for enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome analysis

We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose-bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.     

Production and characterization of cellulase from <Emphasis Type="Italic">E. coli</Emphasis> EgRK2 recombinant based oil palm empty fruit bunch

Oil Palm Empty Fruit Bunch (OPEFB) is an abundant biomass resource in Indonesia, which contains 41.3 ~ 46.5% (w/w) of cellulose. This research examined the production of cellulase by the E. coli EgRK2 recombinant strain using an OPEFB substrate. The production of the enzyme was initially examined to identify optimum growth conditions, by observing the growth and activity of E. coli EgRK2 compared to its wild type. Our results showed that the optimum production time, pH and temperature of the recombinant growth and cellulase activity were achieved at 24 h, and at 7 and 40°C, respectively. Using these optimum conditions, the enzyme was produced, and experiments were carried out to examine the enzyme characteristics, produced from both strains, on hydrolysis of cellulose from OPEFB. Our results showed that the activity of the enzyme produced by the recombinant almost doubled compared to that of the wild type, although the optimum pH for both strains was pH 6. Higher activity was achieved by the recombinant compared to the wild type strain, and values were 1.905 and 1.366 U/mL, respectively. The optimum temperature for hydrolysis by cellulase occurred at 50°C for Bacillus sp. RK2, and 60°C for Bacillus sp. EgRK2. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for OPEFB degradation by E. coli EgRK2 were 0.26% and 1.750 μmol/mL/sec, which were significantly better values than those of the wild type. Control experiments for the degradation test using CMC also showed a better Vmax value for E. coli EgRK2 compared to the wild type, which is 2.543 and 1.605 μmol/mL/sec, respectively.     

Strategies for the Production of Recombinant Protein in Escherichia coli

In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.     

Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from Xenophilus azovorans KF46F

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.     

Microcalorimetric studies on the polyhydroxyalkanoates production of recombinant Escherichia Coli

The thermogenic curves of metabolism of two strains of Escherichia coli pUC19cab/XL-IBlue and XL-IBlue have been determined by using a LKB-2277 bioActivity Monitor and ampoule method at 37°C. pUC19cab/XL-IBlue was a recombinant E. coli strain bearing a foreign plasmid pUC19cab which brought the polyhydroxyalkanoates (PHAs) production. XL-IBlue was a host bacterium without any foreign DNA. Our studies reveal that the PHA production of recombinant E. coli has an apparent influence on their thermogenic curves of metabolism and therefore the initial time of PHAs production can be determined from these thermogenic curves. The text was submitted by the authors in English.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

Cloning and maintenance of the housefly sodium channel gene using low copy number vector and two sequential host strains

In spite of the long history of recombinant DNA technology, some genes have not been successfully cloned in Escherichia coli. This is probably due to the toxic effects of the expressed foreign gene product on E. coli. In initial attempts to clone the full-length Vssc1 voltage-sensitive sodium channel α-subunit gene from houseflies, we used one of the most popular vectors and hosts but were unable to retrieve any intact clone. By using two vectors with different copy numbers and two alternate E. coli host strains, we found that the combined use of a low copy number vector (pALTER-1) and an E. coli host strain that suppresses plasmid replication (ABLE-K) is essential to obtain intact full-length Vssc1 clone. However, since the ABLE-K strain was not a suitable host for the long-term maintenance of Vssc1 gene due to its recombination-positive genotype, it was necessary to transfer the Vssc1 plasmid from the primary host to a secondary host with a recombination-minus genotype (Stbl2) to minimize the chances of deletion or rearrangement. We believe that this cloning strategy, with a low copy number vector and the sequential use of two E. coli strains, will be also applicable for the cloning of other toxic genes.     

Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli

In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l⁻¹.     

The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)

The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).     

Improvement of fatty acid biosynthesis by engineered recombinant Escherichia coli

The purpose of this research was to develop new strains of Escherichia coli with improved fatty acid biosynthesis. β-Ketoacyl acyl carrier protein synthase III (fabH) catalyzes the first step in the synthesis of fatty acids in parallel with acetyl-CoA carboxylase (accABC) and malonyl-CoA: acyl carrier protein transacylase (fabD) in Escherichia coli K-12 MG1655. The enzyme encoded by the fabH gene leads to an increase in the synthesis of short-chain-length fatty acids and a strong preference for acetyl-CoA, as it produces only straight chain fatty acids (SCFAs). It also seems to play a role in determining the type and composition of fatty acids produced. In this study, metabolically engineered strains of E. coli K-12 MG1655 containing fabH or accA::accBC::fabD or accA::accBC:: fabD::fabH gene-inserted expression vector (pTrc99A) were constructed. To observe the effects of overexpression, the production of malonic acid, a pathway intermediate, and fatty acids was analyzed. The resulting recombinant strains produced total lipids up to approximately 1.2 ～ 1.6 fold higher than that of wild-type E. coli. The production of hexadecanoic acid was especially enhanced up to approximately 4.8 fold in E. coli SGJS13 as compared to E. coli SGJS11.     

Molecular and serotyping characterization of shiga toxogenic Escherichia coli associated with food collected from Saudi Arabia

Shiga toxin-producing Escherichia coli (STEC) strains are considered as one of the major food-borne disease agents in humans worldwide. STEC strains, also called verotoxin-producing E. coli strains. The objectives of the present study were serotyping and molecular characterization of shiga toxigenic E. coli associated with raw meat and milk samples collected from Riyadh, Saudi Arabia. A total of 540 milk samples were collected from 5 dairy farms and 150 raw meat samples were collected from different abattoirs located in Riyadh, Saudi Arabia. E. coli were recovered from 86 milk samples (15.93%), serotyping of E. coli isolates revealed, 26 (4.81%) strains O157: H7, 23 (4.26%) strains O111, 20 (3.70%) strains O113: H21, 10 (1.85%) strains O22: H8 and 7 (1.3%) strains O172: H21. Meanwhile, 17 (11.33%) strains of E. coli were recovered from raw meat samples, serotyping of E. coli isolates revealed, 6 (4%) strains O157: H7, 5 (3.33%) strains O111 and 4 (2.67%) strains O174: H2 and only two (1.33%) strains were identified as O22: H8. Shiga toxin2 was detected in 58 (67.44%) serotypes of E. coli recovered from milk samples and 16 (94.12%) serotypes of E. coli recovered from meat samples, while intimin gene was detected in 38 (44.186%) serotypes of E. coli recovered from milk samples and in 10 (58.82%) serotypes of E. coli recovered from meat samples. The results of this study revealed the efficiency of combination between serotyping and molecular typing of E. coli isolates recovered from food of animal origin for rapid detection and characterization of STEC.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].