Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.     

Bacterial polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. More than 80 HAs have been detected as constituents of PHAs, which allows these thermoplastic materials to have various mechanical properties resembling hard crystalline polymer or elastic rubber depending on the incorporated monomer units. Even though PHAs have been recognized as good candidates for biodegradable plastics, their high price compared with conventional plastics has limited their use in a wide range of applications. A number of bacteria including Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, methylotrophs, pseudomonads, and recombinant Escherichia coli have been employed for the production of PHAs, and the productivity of greater than 2 g PHA/L/h has been achieved. Recent advances in understanding metabolism, molecular biology, and genetics of the PHA-synthesizing bacteria and cloning of more than 20 different PHA biosynthesis genes allowed construction of various recombinant strains that were able to synthesize polyesters having different monomer units and/or to accumulate much more polymers. Also, genetically engineered plants harboring the bacterial PHA biosynthesis genes are being developed for the economical production of PHAs. Improvements in fermentation/separation technology and the development of bacterial strains or plants that more efficiently synthesize PHAs will bring the costs down to make PHAs competitive with the conventional plastics. (c) 1996 John Wiley & Sons, Inc.     

Bioproduction of polyhydroxyalkanoates from bacteria: a metabolic approach

The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in Polyhydroxyalkanoates (PHAs) and the biosynthetic machinery for PHA metabolism has been the area of research over the last two decades. PHAs are polyesters of hydroxyalkanoates synthesized by numerous bacterial species with atleast five different PHA biosynthetic pathways. These are accumulated as an intracellular carbon and energy storage material. This diversity, in combination with genetic and molecular engineering has opened up this area for development of optimum PHA producing organisms. Even though PHAs have been recognized as a good candidate for biodegradable plastics, their industrial application is limited owing to high production cost. The classical microbiology and modern molecular biology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHA. This review provides an overview of the different PHA biosynthetic systems, the enzymes involved in PHA biosynthesis and there genetic background followed by a detailed summation of how this natural diversity is being used to develop commercially attractive recombinant process for large scale production of PHAs.     

The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by a wide range of bacteria, including Pseudomonads. These polymers are accumulated in the cytoplasm as carbon and energy storage materials when culture conditions are unbalanced and hence, they have been classically considered to act as sinks for carbon and reducing equivalents when nutrients are limited. Bacteria facing carbon excess and nutrient limitation store the extra carbon as PHAs through the PHA polymerase (PhaC). Thereafter, under starvation conditions, PHA depolymerase (PhaZ) degrades PHA and releases R -hydroxyalkanoic acids, which can be used as carbon and energy sources. To study the influence of a deficient PHA metabolism in the growth of Pseudomonas putida KT2442 we have constructed two mutant strains defective in PHA polymerase ( phaC1 )- and PHA depolymerase ( phaZ )-coding genes respectively. By using these mutants we have demonstrated that PHAs play a fundamental role in balancing the stored carbon/biomass/number of cells as function of carbon availability, suggesting that PHA metabolism allows P. putida to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand. Furthermore, we have established that the coordination of PHA synthesis and mobilization pathways configures a functional PHA turnover cycle in P. putida KT2442. Finally, a new strain able to secrete enantiomerically pure R -hydroxyalkanoic acids to the culture medium during cell growth has been engineering by redirecting the PHA cycle to biopolymer hydrolysis.     

FabG mediates polyhydroxyalkanoate production from both related and nonrelated carbon sources in recombinant Escherichia coli LS5218

Polyhydroxyalkanoates (PHAs) composed of a mixture of short-chain-length-medium-chain-length (SCL-MCL) hydroxyacyl monomers are biologically produced polyesters that have properties ranging from thermoplastic to elastomeric, dependent on the molar ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of properties and applications for SCL-MCL PHA copolymers, it is important to develop and characterize novel metabolic pathways for SCL-MCL PHA production. The current study shows that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes enhances the production of SCL-MCL PHA copolymer from both related and nonrelated carbon sources in Escherichia coli LS5218, indicating the flexibility of FabG as a monomer-supplying enzyme for biological PHA production.     

Preparation of a novel artificial bacterial polyester modified with pendant hydroxyl groups

The Poly(hydroxyalkanoate) (PHA) chemical modifications represent an alternative route to introduce functional groups, which cannot be introduced by bioconversion. PHAs containing unsaturated chains were readily converted into polyesters containing a terminal hydroxyl group on the side chains. With the use of the borane-tetrahydrofuran complex, the pendant side chain alkenes were quantitatively transformed into hydroxyl functions. The conversion proceeded to completion without a significant decrease in molecular weight. The introduction of hydroxyl groups in the products was confirmed from Fourier transform infrared and 1H NMR analysis. The presence of repeating units containing pendant hydroxyl groups in the proportion 25 mol % caused an increase in hydrophilicity of these new PHAs because they were soluble in polar solvents such as ethanol. Besides, these reactive PHAs can be used to bind bio-active molecules or to prepare novel graft copolymers with desired properties.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Preparation and properties of a novel class of polyhydroxyalkanoate copolymers

Polyhydroxyalkanoates (PHAs) are biodegradable aliphatic polyesters, known to be produced by many common microorganisms. Nodax is a recently introduced family of PHA copolymers comprising 3-hydroxybutyrate units and a relatively small amount of other medium chain length 3-hydroxyalkanoate (mcl-3HA) comonomers with side groups of at least three carbon units or more. There are several different grades of copolymers available, depending on the average molecular weight, average mcl-3HA content within the copolymer, and side group chain length of the chosen mcl-3HA unit. PHA copolymers with different mcl-3HA types and contents can be made either by bacterial fermentation or by chemical synthesis. The incorporation of mcl-3HA units into PHAs effectively lowers the crystallinity and T(m) in a manner similar to the effect of alpha-olefins in linear low-density polyethylene. The T(m) can be lowered well below the thermal decomposition temperature of PHAs to make this material much easier to process. The reduced crystallinity provides the ductility and toughness required for many practical applications. The mcl-3HA content regulates the T(m) and crystallinity of copolymer almost independently of the branch size, as long as more than three carbons are present in the side group. On the other hand, the side group chain length of the mcl-3HA has a profound effect on the flexibility of copolymer.     

Polymers of 3-hydroxyacids in plants

Polyhydroxyalkanoates (PHA) are polyesters of bacterial origin that have properties of biodegradable plastics and elastomers. Synthesis of PHA in crop plants would allow the large-scale production and use of these biodegradable and renewable polymers as substitutes for petroleum-derived plastics. Synthesis of a diversity of PHAs in plants, such as Arabidopsis thaliana, rapeseed, corn and cotton, has been demonstrated through the genetic engineering of metabolic pathways in the cytoplasm, plastid and peroxisome. PHA can also be used as a novel tool to study various aspects of plant metabolism, such as the regulation of carbon flux to the fatty acid biosynthetic and degradation pathways.     

Synthesis and characterization of a cationic poly(beta-hydroxyalkanoate)

Poly(beta-hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the carbon source composition in the feed stock, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials. Here, we report the synthesis of the first cationic PHA, poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Pseudomonas putida Gpo1 was used to produce poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-10-undecenoate) (PHOU), whose vinyl-terminated side chains were first converted to terminal epoxides and then modified with diethanolamine. The modification of PHOU was examined using (1)H, COSY, and HSQC NMR and GPC and resulted in a loss of molecular weight due to aminolysis and also in the introduction of side chains terminated with tertiary amine groups, which are protonated at physiological pH. The polycationic PHA is soluble in polar solvents such as DMSO, DMF, and water. The new biodegradable cationic polymers are envisioned as nucleic acid delivery systems.     

Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. The use of mixed cultures and cheap substrates can reduce the production cost of PHA. Accumulation of PHA by mixed cultures occurs under transient conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHA storage and the PHA production rate are dependent on the substrate and the operating conditions used. This work reviews the development of PHA research. Aspects discussed include metabolism and various mechanisms for PHA production by mixed cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on PHA production using mixed cultures; PHA production process design; and characteristics of PHA produced by mixed cultures.     

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.     

Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanoates by Pseudomonas sp. DSY-82

A newly isolated strain, designated as Pseudomonas sp. DSY-82, synthesized medium-chain-length polyhydroxyalkanoate (MCL-PHA) copolyesters when grown on alkanoates from hexanoate to undecanoate as the sole carbon source. When used alone, butyrate and valerate supported the growth of the isolate but not PHA production. However, unusual polyesters containing 3-hydroxyvalerate, as well as various MCL 3-hydroxyalkanoate monomeric units, were synthesized when valerate was cofed with either nonanoate or 10-undecenoate, suggesting the formation of monomer units from both substrates. Concentrations of 3-hydroxyvalerate, 3-hydroxyoctanoate, and 3-hydroxydecanoate in the PHAs produced were significantly elevated by the addition of valerate, indicating that the incorporation of these monomer units to PHA occurred primarily through cometabolism. The total amount of these monomer units in the PHAs reached up to 30%. The PHAs produced in this study were most likely random copolyesters as determined by differential scanning calorimetric analysis. This is the first case of microbial synthesis of copolyesters consisting of 3-hydroxyvalerate and MCL 3-hydroxyalkanoate monomer units through cometabolism.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.     

Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.     

Recent strategies for efficient production of polyhydroxyalkanoates by micro‐organisms

Polyhydroxyalkanoates (PHAs) are polyesters accumulated by many bacteria under unbalanced growth conditions, and have been used to meet the various demands in areas of agriculture, medicine, and materials especially belong to a rapidly expanding area of biomedical research. Unfortunately, the high production cost than the traditional synthetic materials has greatly limited the wide application of PHA. Here, we systematically summarized recent progress in production of PHAs and a series of optimization strategies such as supplying renewable carbon substrates, developing better bacterial strains, optimization of fermentation processes, engineering new pathways and etc., were applied to reduce production cost, therefore providing many new ideas and methods for the production of PHAs in economically viable processes. This is believed to be a comprehensive report to show different strategies and methods for low‐cost production of PHAs. Further studies are still needed to make PHAs more and more economically viable to meet a wide range of applicability.     

聚羟基脂肪酸酯纳米微球：结构特征、生物合成及其在生物技术和生物医药领域的应用

聚羟基脂肪酸酯（polyhydroxyalkanoate）PHA 纳米微球是很多微生物在营养失衡的情况下,在体内合成的一种可生物降解的细胞内聚酯,主要作为微生物的碳源及能量储备。天然 PHA 微球的内部是由疏水的聚酯链构成的疏水核心,其外层是由磷脂界膜及膜上嵌入或附着的包括 PHA合酶 PhaC 和 PHA 颗粒相关蛋白 PhaP 等蛋白构成的边界层。PhaC 通过共价键连接在PHA微球表面,而 PhaP 通过疏水相互作用吸附在 PHA 微球表面。通过将外源性功能蛋白与 PhaC 或 PhaP 进行融合表达,在重组微生物体内就能直接合成表面带有功能蛋白的纳米微球复合体。由于该纳米微球在微生物细胞内是以独立的包涵体形式存在,因此通过细胞破碎及离心等方法就能简便、有效地使其从细胞中分离并得以纯化。鉴于 PHA 微球这种表面易被修饰改造的特性,越来越多的功能蛋白通过与 PHA 微球表面蛋白（PhaC 或 PhaP）的融合表达,呈递在了 PHA 微球表面,使其成为一种廉价、高效的蛋白固定化及呈递的新技术。本文在介绍了 PHA 微球的结构特性及生物合成的基础上,着重综述了目前关于功能化 PHA 微球在蛋白纯化、固定化酶、生物分离、靶向递药、疾病诊断、成像技术及新型疫苗开发方面的研究现状及其未来在生物医药等领域的广泛应用前景。     

Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.

The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.