Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Construction of recombinant Escherichia coli strais for production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate)

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC 19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.     

Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

The present study focuses on predicting the concentration of intracellular storage polymers in enhanced biological phosphorus removal (EBPR) systems. For that purpose, quantitative image analysis techniques were developed for determining the intracellular concentrations of PHA (PHB and PHV) with Nile blue and glycogen with aniline blue staining. Partial least squares (PLS) were used to predict the standard analytical values of these polymers by the proposed methodology. Identification of the aerobic and anaerobic stages proved to be crucial for improving the assessment of PHA, PHB and PHV intracellular concentrations. Current Nile blue based methodology can be seen as a feasible starting point for further enhancement. Glycogen detection based on the developed aniline blue staining methodology combined with the image analysis data proved to be a promising technique, toward the elimination of the need for analytical off-line measurements.     

Medical applications of biopolyesters polyhydroxyalkanoates

Microbial polyhydroxyalkanoates(PHAs) are a family of biopolyesters produced by many wild type and engineered bacteria.PHAs have diverse structures accompanied by flexible thermal and mechanical properties.Combined with their in vitro biodegradation,cell and tissue compatibility,PHAs have been studied for medical applications,especially medical implants applications,including heart valve tissue engineering,vascular tissue engineering,bone tissue engineering,cartilage tissue engineering,nerve conduit tissue engineering as well as esophagus tissue engineering.Most studies have been conducted in the authors’ lab in the past 20+ years.Recently,mechanism on PHA promoted tissue regeneration was revealed to relate to cell responses to PHA biodegradation products and cell-material interactions mediated by microRNA.Very importantly,PHA implants were found not to cause carcinogenesis during long-term implantation.Thus,PHAs should have a bright future in biomedical areas.     

Degradation of commercially important polyhydroxyalkanoates in tropical mangrove ecosystem

This paper presents the degradation trends of selected polyhydroxyalkanoate (PHA) films in a tropical mangrove environment. The biodegradability of homopolymer poly(3-hydroxybutyrate) [P(3HB)] and its co-polymers, poly(3-hydroxybutyrate-co-5 mol% 3-hydroxyvalerate) [P(3HB-co-5 mol% 3HV)] and poly(3-hydroxybutyrate-co-5 mol% 3-hydroxyhexanoate) [P(3HB-co-5 mol% 3HHx)], was investigated along with P(3HB) films containing 38 wt% titanium dioxide (TiO₂) [P(3HB)-38 wt% TiO₂]. The degradation of these formulations was monitored for 8 weeks at three different zones in an intermediate mangrove compartment along Sungai Pinang, adjacent to a famous fishing village on south of Penang Island. The degradation rate was observed both on the surface and in the sediment and was expressed in percentage of weight loss. The microbial enumeration done using sediment from the different zones indicated similar colony-forming unit (CFU) counts even though differences were noticed in the degradation profile of the various films in the respective zones. The results obtained revealed that co-polymers disintegrated at similar or higher rate than the homopolymer, P(3HB). However, the incorporation of TiO₂ into PHB films caused the degradation rate of P(3HB)-38 wt% TiO₂ composite film to be far slower than all the other PHA films. The overall rate of degradation of all PHA films placed on the sediment surface was slower than those buried in the sediment. Microscopic analyses showed that the surface morphology of P(3HB-co-5 mol% 3HHx) was more porous compared to P(3HB) and P(3HB-co-5 mol% 3HV) films, which may be an important factor for its rapid degradation.     

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer from wild-type Comamonas sp. EB172

Poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer was produced by Comamonas sp. EB172 using single and mixture of carbon sources. Poly(3-hydroxyvalerate) P(3HV) incorporation in the copolymer was obtained when propionic and valeric acid was used as precursors. Incorporation of 3HV fractions in the copolymer varied from 45 to 86 mol% when initial pH of the medium was regulated. In fed-batch cultivation, organic acids derived from anaerobically treated palm oil mill effluent (POME) were shown to be suitable carbon sources for polyhydroxyalkanoate (PHA) production by Comamonas sp. EB172. Number average molecular weight (M_n) produced by the strain was in the range of 153-412 kDa with polydispersity index (M_w/M_n) in the range of 2.2-2.6, respectively. Incorporation of higher 3HV units improved the thermal stability of P(3HB-co-3HV) copolymer. Thus the newly isolated bacterium Comamonas sp. EB172 is a suitable candidate for PHA production using POME as renewable and alternative cheap raw materials.     

Separation of poly-3-hydroxyvalerate-co-3-hydroxybutyrate through gradient polymer elution chromatography

Polyhydroxyalkanoates are biodegradable polyesters produced by bacteria that can have a wide distribution in molecular weight, composition of monomers, and functionalities. This large distribution often leads to unpredictable physical properties making commercial applications challenging. To improve polymer homogeneity and obtain samples with a clear set of physical characteristics, poly-3-hydroxyvalerate-co-3-hydroxybutyrate copolymers were fractionated using gradient polymer elution chromatography (GPEC) as opposed to extensively used bulk fractionation. Separation was achieved using a reversed-phase column with chloroform and ethanol as the solvent and non-solvent, respectively. A separation was also conducted on a normal-phase column to compare elution patterns between columns of varied polarity. The fractions were analyzed using Size Exclusion Chromatography (SEC) and NMR to determine the percentage of 3-hydroxyvalerate in the copolymer as well as its molecular weight. It was found that as the percentage of "good" solvent was increased in the mobile phase, the polymers eluted with decreasing percentage of 3-hydroxyvalerate and increasing molecular weight which indicates the importance of precipitation/redissolution in the separation. The elution pattern of the polymer remained unchanged when using both a normal- and reversed-phase column which also illustrates the dominance of precipitation/redissolution in GPEC of polyhydroxyalkanoates. As such, GPEC is shown to be an excellent choice to provide polyhydroxyalkanoate samples with a narrower distribution in composition than the original bulk copolymer sample.     

以葡萄糖为碳源合成生物降解性聚酯的研究

利用从油田土壤中筛选的菌种ＤＧ１７ 以葡萄糖为碳源通过微生物发酵法合成了具有不同结构单元的新型生物可降解性聚合物———聚羟基脂肪酸酯（ＰＨＡｓ） ．初步研究了ＤＧ１７ 以葡萄糖为碳源的生物合成规律,并借助ＧＣ、ＮＭＲ 等分析手段对合成的聚合物进行了结构的分析表征,另外还研究了ＰＨＡｓ 的活性污泥降解情况．研究表明,在限氮条件下,只有碳氮比高于５后,ＤＧ１７ 才能在其体内合成ＰＨＡｓ．在过量碳源的存在下,氮磷比低,得到的聚合物是一种具长侧链的聚（ 羟基辛酸 ｃｏ 羟基癸酸） 的共聚物,为一种热塑性弹性体．在硫酸铵浓度为０－５ｇ／Ｌ,碳氮比为２０ 条件下合成的Ｐ（ＨＯ ｃｏ ＨＤ） 热塑性弹性体的数均分子量为１－１６ ×１０ － ５ ,分子量分散指数为２－４３ ．其玻璃化温度及熔融温度分别为Ｔｇ ＝ － ５２ ℃,Ｔｍ ＝ ５０ ℃．氮磷比高,则合成热塑性塑料ＰＨＢ．结果表明培养基中氮源与磷酸盐的相对浓度是影响ＤＧ１７ 生物合成路径的重要条件．     

Effect of C∶N molar ratio on monomer composition of polyhdroxyalk anoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria. In this study, the effect of C∶N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkan⇘te produced by Pseudomonas pseudoalkaligenus YS1. It was observed that there were some fixed ranges of C∶N molar ratio that affect the monomer composition of PHA independently of the substrate. For strain 0806, the ranges were C∶N <20, 20<C∶N<200, and C∶N>200. The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate. For strain YS1, the ranges were C∶N<20, 20<C∶N<45, and C∶N>45. These results are useful for controlling monomer composition in PHA production.     

Polyhydroxybutyrate production from carbon dioxide by cyanobacteria

Genetic characterization and enhancement of polyhydroxybutyrate (PHB) accumulation in cyanobacteria were investigated for efficient PHB production from CO₂. The genome DNAs in the PHB-accumulating strains Synechococcus sp. MA19 and Spirulina platensis NIES46 retained the highly homologous region to phaC of Synechocystis PCC6803, whereas low homology was detected in the nonaccumulating strains Synechococcus sp. PCC7942 and Anabaenacylindrica NIES19. Synechococcus sp. MA19, which accumulates PHB up to 30% of dry cell weight from CO₂ as the sole carbon source, was mutated by insertion of transposon Tn5 to enhance the PHB accumulation. Genetic and physiological analysis of the mutant indicated that decreased phosphotransacetylase activity could trigger an increase of acetyl coenzyme A leading to enhancement of PHB accumulation. PHB synthase in Synechococcus sp. MA19 was probably attached to thylakoid membrane since PHB granules were associated with pigments. A genetically engineered cyanobacteria retaining soluble PHB synthase from Ralstonia eutropha accumulated pigment-free PHB granules, which is an advantage for the purification of PHB.