Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene
- Xue Gao†1,
- Xiao-Xi Yuan†1,
- Zhen-Yu Shi2,
- Ying-Ying Guo1,
- Xiao-Wen Shen1,
- Jin-Chun Chen1,
- Qiong Wu1Email author and
- Guo-Qiang Chen1, 3Email author
© Gao et al.; licensee BioMed Central Ltd. 2012
Received: 30 June 2012
Accepted: 12 September 2012
Published: 14 September 2012
Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield.
To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2 Ps O with a hairpin structure in the 5’ untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2 Ps .
The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.
Polyhydroxyalkanoates (PHA) are a family of biopolyesters accumulated by many bacteria, and have been studied to meet the various demands in chemical, material and special industries [1–6]. The structures and properties of PHA can be adjusted by controlling their monomer compositions [7, 8]. On the basis of the carbon chain lengths of monomers, PHA are classified as short-chain-length PHA (scl PHA) and medium-chain-length PHA (mcl PHA) consisting of three to five and six or more carbon atoms in their monomers, respectively [7, 9]. The differences in PHA monomers and compositions can strongly affect the properties and qualities of the polyesters [10–12]. Generally, PHA copolymers consisting of scl and mcl monomers are considered to have more potentials for applications due to their suitable properties including tensile strength, Young’s modulus, elongation at break and so on . Particularly, the scl and mcl PHA copolymers consisting of high molar percentage of scl monomers (such as 3HB) and low molar percentage of mcl monomers have better application properties including high melting temperature and toughness as well as rapid crystallization process . Many bacteria such as Aeromonas hydrophila and Ralstonia eutropha were reported to be able to produce scl and mcl PHA with different compositions and yields [15, 16]. However, there are still difficulties to control the PHA monomer compositions when industrial fermentation processes are conducted for production of PHA .
One of the key factors that determine PHA monomers composition is the substrate specificity of the PHA synthase (PhaC). The production of scl and mcl PHA requires synthases with relatively wide substrate specificities, such as PhaC from Aeromonas caviae[17, 18] and PhaC2Ps from Pseudomonas stutzeri 1317 (named PhaC2Ps) . These PHA synthases have been well characterized and expressed for production of scl and mcl copolymers [17, 19]. For example, when expressed in a PHA synthase gene phbCRe negative mutant R. eutropha PHB-4, PhaC2Ps could confer on the host strain the ability to synthesize PHA with monomer compositions adjustable by altering carbon sources . Furthermore, to achieve a high mole faction of 3-hydroxybutyrate (3HB) monomer in scl-mcl PHA copolymers for better thermal and mechanical properties, specific point mutagenesis was successfully applied , leading to mutated PHA synthase PhaC2PsQKST that increased PHA accumulation up to 42 wt% consisting of 93 mol% 3HB in R. eutropha PHB-4. However, all of the PhaC2Ps mutants have not yet been characterized in E. coli which is a well-developed cell factory for many fine products .
E. coli was successfully exploited for scl PHA  and scl-mcl PHA production [23, 24]. Particularly, pathways were constructed to achieve scl-mcl PHA accumulation in E. coli using related or unrelated carbon sources [25–27]. However, the overall PHA yields were generally low probably due to inadequate supply of PHA precursors, and/or lower synthase activity during the polymerization process.
The stability of mRNA is one of the most significant factors that affect levels of protein synthesis, in this case, PHA synthase synthesis. It was reported that the secondary structures within 5’ untranslated regions (UTRs) of prokaryotic mRNA could act as mRNA stabilizers, prevent them from fast degradation by RNases and therefore promote translation . Various rationally designed synthetic 5’ hairpin structures have been investigated and their half lives and effects in mRNA stability were measured [28, 29], and it is significant to add different mRNA secondary structures in the 5’ UTR region for controlling the expression of recombinant genes. On the other hand, species-specific variations in codon usage are generally considered one of the major factors that affect heterologous protein expressions. If the concentrations of the E. coli tRNAs for the rare codons are insufficient to optimally translate mRNA , codon optimization could be effective to enhance protein expression [31, 32].
To achieve high scl-mcl PHA production, the functions of the wild type phaC2 Ps and its recombinants in E. coli were evaluated in this study, and codon optimization and mRNA stabilization strategy were employed to enhance PHA synthase expression. The recombinant E. coli expressing the optimized PhaC2Ps was proven to be able to produce scl-mcl PHA much more effectively than the wild type did.
Analysis and optimization of the mutated PHA synthase PhaC2PsQKST
To further enhance the expression level of the target protein, the hairpin structure pHP17 (Figure 2), which was reported to have the longest half life among a hairpin mRNA pool , was chosen to evaluate its effect on the expression of PhaC2PsQKST. To do this, pHP17 was inserted to the upstream of the gene phaC2 Ps O, resulting in phaC2 Ps OH [GenBank: JX082171].
Effect of the optimization strategies on mRNA degradation and protein expression of phaC2 Ps
To assess the effect of codon optimization and hairpin stabilization, five pET28a-based plasmids were constructed expressing different phaC2 Ps genes, namely pETC2, pETC2QK, pETC2QKST, pETC2O, and pETC2OH. To demonstrate the effect of the hairpin introduction, the decay profiles of the optimized PHA synthase mRNAs with or without the hairpin structure were determined via quantitative real-time PCR. Figure 1a showed the percentages of the remaining mRNA at different time intervals. The mRNA with the hairpin structure (expressed in pETC2OH) appeared to be more stable than the one without it (expressed in pETC2O), indicating the hairpin structure had contributed to an enhanced level of gene expression.
The PHA synthase expression levels were further examined by western blot analysis (Figure 1b). While the original PhaC2Ps, the mutants PhaC2PsQK and PhaC2PsQKST were expressed with similar abundance, codon optimization of the gene significantly increased the protein expression level (lane 5). With hairpin structure inserted in front of the gene, the protein expression was further enhanced (lane 6).
PHB accumulation in recombinant E. coli expressing optimized PhaC2Ps
Bacterial strains and plasmids
Strains or plasmids
Source or reference
E. coli JM109
recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac proAB)/F’ [traD36, proAB + , lac q lacZΔ M15]
TaKaRa (Dalian, China)
E. coli LS5218
pBluscript SK- derivative, phbA Re and phbB Re cloned from R. eutropha
pBluscript SK- derivative, phbA Re and phbB Re cloned from R. eutropha phaC2 Ps cloned from P. stutzeri 1317
pBBR1MCS-2 derivative, mutated phaC2 Ps Gln482→Lys (CAG→AAG)
pBBR1MCS-2 derivative, double mutated phaC2 Ps Ser326→Thr (AGC→ACC) Gln482→Lys (CAG→AAG)
Wild type phaC2 Ps inserted into pET28a
Mutated phaC2 Ps inserted into pET28a
Double mutated phaC2 Ps inserted into pET28a
Codon optimized phaC2 Ps QKST inserted into pET28a
Codon optimized phaC2 Ps QKST with hairpin sequence inserted into pET28a
pBHR69 derivative, wild type phaC2 Ps of P. stutzeri 1317, plus phbA Re and phbB Re
Mutated phaC2 Ps inserted into pBHR69
Double mutated phaC2 Ps inserted into pBHR69
Codon optimized phaC2 Ps QKST inserted into pBHR69
Codon optimized phaC2 Ps QKST with hairpin sequence inserted into pBHR69
Scl-mcl PHA production by recombinant E. coli
Scl and mcl PHA production by E. coli LS5218 harboring different PhaC2 Ps s a
CDW b (g/l)
PHA content c (wt%)
PHA composition (mol %) e
Scl and mcl PHA accumulation by recombinant E. coli LS5218 harboring pYC2OH grown in mixtures of gluconate and dodecanoate a
Concentration ofgluconate (g/l)
PHA content (wt%)
PHA composition (mol %)
With the development of synthetic biology, many functional modules are designed to be heterologously expressed in a host strain, and thus efficient and sometimes precise expression of a specific enzyme is generally required to meet the demand of coordination among multiple modules . There are many possibilities to adjust expression levels of proteins in different situations, and codon optimization is generally considered a universal means to control or enhance heterologous protein expression [31, 32]. Recent development in DNA synthesis and sequencing allows the rapid and accurate synthesis of large amount of DNA constructs at a lower cost, providing more accessibility to optimizing codon usage of heterologously expressed genes and to screening for suitable expression levels . The “one amino acid-one codon” optimization strategy was employed in this study. PHB accumulation studies indicated that the mutagenesis and optimization of PHA synthase PhaC2Ps cloned from P. stutzeri 1317 significantly enhanced the synthase polymerization activity, and thus promoted PHB synthesis (Figure 3). It was previously reported that PhaC2Ps could achieve up to 40 wt% PHB accumulation when expressed in R. eutropha PHB-4 [19, 20]. However, when expressed in E. coli, this PHA synthase could only accumulate trace amount of PHB (Figure 3). The series of site-specific mutagenesis of this synthase PhaC2Ps was reported to achieve higher PHA accumulation and 3HB fraction in R. eutropha PHB-4 , and the same phenomenon could be observed when the mutated enzymes were expressed in E. coli (Figure 3). The double mutant PhaC2PsQKST could enhance PHB accumulation approximately 6-fold compared to the wild type did, and this was partially due to the altered substrate-specificity . Another 1.7-fold of PHB accumulation was observed in the case of codon-optimized synthase PhaC2PsO (Figure 3). Since the specificity was not altered compared to PhaC2PsQKST (the amino acids sequence was exactly the same), this result indicated that the increased PHB content was completely due to the enhancement of synthase expression.
The FadR-deficient strain E. coli LS5218 was used as the host to evaluate the scl-mcl PHA production driven by different PhaC2Pss. When grown on MS medium supplemented with 8 g/l dodecanoate, all five recombinants harboring different plasmids could accumulate scl-mcl PHA (Table 2). Yet for strains harboring pYC2 or pYC2QK, only 3HB, 3HO and 3HD monomers were detected. Compared to the wild type PhaC2Ps, the two site-specific mutants PhaC2PsQK and PhaC2PsQKST increased PHA content and thus also the cell dry weight. Simultaneously, the substrate specificity was altered as a higher fraction of 3HB was observed, which was consistent with the previous report . However, the overall PHA content was relatively low (1.79 wt%). After codon optimization of phaC2 Ps QKST, the PHA content was significantly increased to 3.40 wt% and various PHA monomers were found including 3HB, 3HHx, 3HO, 3HD and 3HDD. Particularly, the 3HB monomer fraction was reduced slightly compared to the situation of PHA synthesis by PhaC2PsQKST (Table 2), indicating that the monomer composition relies on multiple factors including the substrate specificity, the polymerization efficiency and the precursor supply. Both the PHB and scl-mcl PHA productions by the optimized PhaC2PsQKST verified that the codon optimization strategy worked well in this condition (Figure 3 and Table 2). The replacement of rare codons such as AGG (Arg) and CUA (Leu) led to an significant increase in PHA production, indicating that the expression level of the key enzyme PHA synthase was strongly enhanced. It should be mentioned that there are other algorithms for codon optimization, and their functions on this specific case remain to be explored. It is expected that further optimization on PHA synthases can lead to wider substrate specificity and higher expression level, allowing the precise control of PHA polymerization.
Altering expression related factors such as promoters, ribosome binding sites, and mRNA stabilizers are possibilities to change the transcription and translation level of key PHA synthesis enzymes. In this study, a hairpin structure pHP17 which was reported to increase the half life of mRNA up to 19.8 min  was inserted in the 5’ UTR of phaC2 Ps O, and it contributed another 1.5-fold in PHB accumulation (Figure 3), demonstrating that the degradation of the mRNA of phaC2 Ps is a significant factor that affects PHA accumulation. In the case of scl-mcl PHA production, the contribution of the hairpin structure was also significant (p < 0.01). However, since the main function of this structure is to increase mRNA stability, it is possible that the effect of a hairpin structure is largely dependent on the expression profiles in different conditions.
For the last version of the optimized PhaC2Ps (codon optimized and hairpin inserted) the effect of different precursor supply situations to the monomer compositions of scl-mcl PHA was investigated. Compared to the presence of 20 g/l gluconate along with 8 g/l dodecanoate (Table 3), E. coli LS5218 harboring pYC2OH could accumulate slightly more PHA when 5 g/l gluconate or 10 g/l were added, and the CDW was also higher. This was probably due to a better balance that could be reached between cell growth and PHA accumulation under low gluconate concentration, which allowed proper coordination with the specificity and expression level of the PHA synthase. On the other hand, monomer compositions can be adjusted by providing different ratios of precursors coming from multiple metabolism pathways. Since E. coli has been well investigated and is relatively easy for genetic integration, we can expect efficient scl-mcl PHA production from unrelated carbon sources if precursor supplying pathways are introduced, and precise control of the carbon flux through these pathways would lead to PHA production with desired compositions. Furthermore, expression cassettes with high efficiency is often required for chromosomal expression since it is generally more difficult compared to plasmid system, therefore this investigation provides an optimized PHA synthase which may contribute to plasmid-free PHA production in E. coli in the future.
In summary, a series of optimization strategies were applied on the PHA synthase PhaC2Ps from P. stutzeri 1317, which is a PHA synthase with wide substrate specificities, to optimize its heterologous expression and PHA production in E. coli. The codon optimization and hairpin structure in the 5’-UTR of the mRNA were effective for optimizing PhaC2Ps expression, and both the PHB and scl-mcl PHA production levels were significantly increased. The results indicated that these strategies provide a good opportunity for future PHA production with optimized yield and composition.
Optimization of the coding sequence and transcription region of target PhaC gene
For codon bias optimization, “one amino acid-one codon” strategy  was employed in this study to obtain an optimized translational region of the target gene. Codon preference in E. coli K-12 was selected and the codons of predicted highly expressed genes were chosen to replace rare ones. A hairpin structure which was reported to be able to increase mRNA stability  was introduced in the 5’ UTR of the mRNA for the target gene. The entire coding sequence of the optimized gene along with the hairpin region [GenBank: JX082171] was synthesized by Shanghai Qinglan Biotech Co., Ltd.
Bacterial strains, plasmids, and general procedures for DNA manipulation
The main bacterial strains and plasmids used in this study are listed in Table 1. E. coli Trans1-T1 (Transgen) strain was used for plasmids construction mentioned in this study. E. coli JM109 and E. coli LS5218  were used for evaluating the expression of the PHA synthase PhaC2Ps and accumulating PHA with different compositions.
Primers for construction and verification of plasmids
5’- ATTGAGCTC GTATTTTGGATGATAACGAGGAGGTAAATAATGCGAGACAAGCCCAATAG a
5’- AATTACTAGT CCTCAGCGGATATGCACGTAGG -3’
5’- ATAATTGAGCTC CACGTCGACTTATCTCGAGACTG -3’
5’- ATAATTGAGCTC GTATTTTGGATGATAACGAGGAG -3’
5’- ATAATTACTAGT CCTTAACGGATGTGAACGTAGGT -3’
5’- ATTCTGTGGATAACCGTATTACC -3’
5’- GCACACCTTGTTGATGGTCATGG -3’
Analysis of the mRNA decay profiles by quantitative real-time PCR
The recombinant cells (E. coli JM109 harboring pETC2O or pETC2OH) were cultured in LB medium. When OD600 reached 0.2, isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.1 mol/l) were added and cells were cultured for another 2 h. Rifampicin was added to a final concentration of 50 μg/ml to inhibit RNA synthesis. After 1 min of incubation, culture samples were iced at timed intervals. Total RNA was isolated from each sample using the E.Z.N.A.® Bacterial RNA Kit (Omega Bio-tek). The purity and concentration of RNA were checked using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Following cDNA synthesis was performed using the Quantscript RT Kit (Tiangen).
The mRNA decay profiles were determined by real-time PCR using the cDNA samples as the templates. The ompA gene was selected as the housekeeping gene. The real-time PCRs were performed using the SuperReal PreMix (SYBR Green) kit (Tiangen) with MxPro 3000P (Agilent Technology). The primers for real-time PCR are listed in Table 1, and the reaction condition was as follows: 95°C for 5 min; 95°C for 20 s, 55°C for 30 s, and 72°C for 30 s for 40 cycles.
Western blot analysis
The recombinant strains were cultivated in LB medium with IPTG (0.1 mol/l) and then harvested by centrifugation. After resuspending the cells in PBS buffer, crude extracts were obtained by disrupting the cells with sonication. The concentrations of total proteins were determined with a BCA protein assay kit (Vigorous). Proteins (10 μg) in crude extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis of PhaC2Ps was carried out using anti His-tag mouse monoclonal antibodies (CW Biotech). Horseradish peroxidase (HRP)-conjugated anti-mouse antibodies (Santa Cruz Biotechnology) were used as a secondary antibody.
Medium and culture conditions
Different strains of E. coli were grown in LB medium or on LB agar plates at 37°C. Antibiotics including ampicilin (100 mg/ml) and kanamycin (50 mg/ml) were added when necessary. Antibiotics were purchased from Sigma (Beijing, China). For PHA accumulation, shake flask experiments were carried out in a rotary shaker (NBS, Series 25D, New Brunswick, NJ, USA) at 200 rpm and 37°C using 500-ml conical flasks each containing 50 ml of medium. 20 g/l gluconate was used for the evaluation of optimized PhaC2Ps for enhanced poly-3-hydroxybutyrate (PHB) synthesis, and 8 g/l dodecanoate plus 0 g/l, 5 g/l, 10 g/l, or 20 g/l gluconate was added as carbon sources in the case of scl-mcl PHA production. To achieve better utilization of dodecanoate, the flasks were shaken at 60 rpm, 45 °C immediately after sterilization in a rotary shaker until dodecanoate was scattered. Cells of E. coli JM109 or LS5218 were first cultured in LB medium for 12 h and then transferred to mineral salt (MS) medium containing different carbon sources at an inoculation volume of 4% for 48 h. The MS medium contained (per liter) 9.0 g Na2HPO4·12H2O, 1.5 g KH2PO4, 1.0 g (NH4)2SO4, 0.4 g MgSO4·7H2O and 2% (v/v) trace element solution as described in previous studies . 100 mg/l thiamine was added into the MS medium when E. coli JM109 was used since this strain is thiamine-deficient. For scl-mcl PHA production, 1 g/l yeast extract was added for the culture of E. coli LS5218. IPTG (0.1 mol/l) was used as an inducing agent for lac promoter and was added at 6 h after inoculation.
Gas chromatography (GC) analysis of PHA in dry cells
Cell dry weight (CDW) was evaluated by harvesting 30 ml cell cultures, centrifuged at 10,000 rpm for 10 min and then washed with distilled water when gluconate was added as sole carbon source. In the case of scl-mcl PHA production using dodecanoate as a carbon source, cells were washed twice with ethanol before washed with distilled water. CDW was measured after vacuum lyophilization (free of the remaining C12 carbon sources). The lyophilized cells and standard PHA samples were subjected to methanolysis in chloroform at 100°C for 4 h in the presence of 3% (v/v) H2SO4 to prepare samples for gas chromatography (GC) (SHIMADZU GC-2014C, Kyoto, Japan) equipped with 30-m HP-5 capillary column . PHA contents were determined by analyzing the GC data.
For statistical analysis, three parallel experiments were conducted and data collected were evaluated by mean ± SD. Student’s two-tailed t-test was performed to determine the significance of differences (**: p < 0.01, *: p < 0.05).
This research was financially supported by the 973 Basic Research Fund (Grant No. 2012CB725201 to GQC and Grant No. 2012CB725204 to QW), the National High Tech Suporting Grants (Project No. 2012BAD32B02 to JCC) and the National Natural Science Foundation of China (Grant No. 31170099 and Grant No. 31170940). We are grateful to Dr. A. Steinbüchel of the University of Münster, Germany, for the generous donation of the plasmid pBHR69.
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