Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 193-199

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 193-199  

Biosynthesis of Polyhydroxyalkanoates*

CHEN Guoqiang ** , WU Qiong , CHEN Jinchun

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

* Supported by the Major Research Project of the Ninth-Five Plan (1996-2000) of China (No.96-C03-03-02), Tsinghua University “985” Project
 ** To  whom correspondence should be addressed

Received: 2000-11-30

Code Number: ts01065

Abstract:   

Many bacteria are able to synthesize polyhydroxyalkanoates, abbreviated as PHA, as carbon and energy storage compounds. PHA with over 90 different monomer structures has been reported and the number keeps increasing. The piezoelectricity, non-linear optical activity, biocompatibility and biodegradability of PHA have offered promising applications in areas such as tissue engineering, drug delivery, smart materials and degradable packaging. Various microorganisms obtained from nature, genetic engineering and mutations have been used for microbial production of PHA as biodegradable plastics. In fact, PHA synthesizing bacteria can be easily found in various locations, especially oil-contaminated soils. Many bacterial strains possessing the ability to synthesize PHA with various monomers have been isolated from oil-contaminated soils. In molasses-contaminated soil, 40% of the isolated bacteria were capable of growing rapidly and synthesizing PHA at the same time. It seems that nature has provided endless numbers of novel PHA synthesizing strains.

Key  words:  polyhydroxybutyrate; PHB; polyhydroxyalkanoates (PHA); biopolymer; polyester

Introduction   

Polyhydroxyalkanoates (PHA) is a family of intracellular biopolymers synthesized by many bacteria as intracellular carbon and energy storage granules (Figs.1 and 2). PHA is normally synthesized under conditions of restricted growth[1-6]. Industrial production has been used to produce PHAs that are environmentally degradable thermoplastics[7-10].

Poly -3-hydroxybutyrate (PHB), the most commonly found member of the PHA family, is a short chain length PHA (scl PHA) with monomers containing 4-5 carbon atoms. Other PHA containing monomers having 6-16 carbon atoms have been termed as medium chain length PHA (mcl PHA)[3]. Copolyesters of PHA containing scl monomers such as 3-hydroxybutyrate (HB) and mcl monomers such as 3-hydroxyhexanoate (HHx) have dramatically improved mechanical properties compared with PHB[11] . Many efforts have been made to screen microorganisms capable of synthesizing PHA copolymers consisting of HB and mcl HA monomers. However, only a few wild type microorganisms and recombinant bacteria have been reported to be able to produce HB and mcl HA containing PHA, regardless of copolymers or blend polymers[11-15].

Many structural variations of PHA have also been synthesized, but the small amount of these unconventional PHA have produced only a few physical characterizations and a few applied results so far[16-19]. Recent PHA research has been directed towards the design, biosynthesis, and properties of biodegradable and biocompatible materials which can be used for bioengineering new optical and other “smart” chiral materials[18].

The review introduces recent advances in this area. Many of these were obtained in our lab, some of them still unpublished data. It is hoped that academic and industrial communities, especially biologists, polymer scientists and material scientists can collaborate closely to explore novel applications of these unique polyesters. In addition, these polymers can only be thoroughly investigated if sufficient PHA is available for studies.

1 Microbial Synthesis of PHA Is a Common Phenomenon

Many bacteria have been reported to accumulate PHA from various substrates such as glucose, sucrose, n-alkanes, n-alcohols or n-organic acids[1, 3, 5, 20]. There has been no systematic study concerning PHA accumulation by microorganisms grown in various geological locations, as it is difficult to carry out large scale PHA analysis using gas chromatography[21]. Recently, our lab developed a rapid noninvasive technique using Fourier Transformed Infrared Spectroscopy (FT-IR) which allows us to detect PHA accumulation intracellularly within a few seconds[22]. If the PHA content is high in the cells, the FTIR spectra of PHA can also differentiate the various structures of PHB, mcl PHA and PHA consisting of HB and mcl HA monomers[22]. Among the PHA, PHB appears to be the most common intracellular storage compound occurring in all locations. Results have shown that mcl PHA, as well as PHA consisting of HB and mcl HA are commonly synthesized by bacteria in oil-contaminated soils or waters[23]. Depending on the substrates, bacteria may or may not synthesize PHA.A total of 371 strains isolated from 20 soil samples collected from various locations showed that approximately 40% of the strains had PHA accumulation ability from at least one carbon sources used in our screening study (Fig. 2). Considering that many bacteria may be able to synthesize PHA from other carbon sources besides the six employed in this study, the actual number of PHA producing bacteria in oil-contaminated soil may be much greater.Our screening work demonstrated that many bacteria possess the ability to synthesize PHA, provided a suitable substrate is available. This result contradicts the generally believed argument that PHA is synthesized when growth conditions are unbalanced, such as nitrogen, phosphate or oxygen limitations or an oversupply of carbon[1].

A number of the strains were shown to have PHA characteristic bands around 1735 cm^-1  on FT-IR spectra when grown on glucose or molasses substrates, demonstrating the possible existence of HB and mcl HA containing PHA in these bacterial cells[22]. Since copolymers of HB and mcl HA have improved mechanical properties and processability, they are more interesting for industrial applications as biodegradable materials[11]. Among the 19 strains studied, 14 strains accumulated PHA containing HB and mcl HA monomers although the PHA were found to be blend polymers consisting of HB and mcl HA (unpublished data). It seemed that these bacteria contained two PHA synthase systems that are responsible for synthesizing PHB and mcl PHA[24]. Therefore, oil-contaminated soils may in general contain many bacteria that are capable of synthesizing PHB, mcl PHA, and PHA consisting of HB and mcl PHA. Copolymers of HB and mcl HA may also be found among oil-contaminated soil, but further screening and studies are needed.

2 Microbial Synthesis of PHA Consisting of Diversified Monomers

The monomer structures of PHA are normally very dependent on the substrates used for cell growth. For example, when Pseudomonas aeruginosa 44T1 was grown on euphorbia oil, the novel monomers in Fig.3 structures (a) and (b) were identified in the PHA[17]. A mixture of octanoate and para-cyanophenoxyhexanoate produced a PHA copolymer consisting of related monomer structures (Fig.3 structure (c))[17]. On soybean oil, a unique monomer tructure was synthesized by strain Pseudomonas stutzeri1317 (Fig.3 structure (d))[18]. Pseudomonas oleovorans grown on 1-heptanene or on a mixture of octane and 1-chlorooctane synthesized monomers of PHA with unsaturated bonds or with chlorine at the end of the side chains (Fig.3 structures (e) and (f))[21, 22]. Various monomer structures of PHA were synthesized by Pseudomonas putida grown on a mixture of octanoate and paracyano-phenoxyhexanoate, on phenoxyalkanoates, on a mixture of nonanoic acid and 10-undecynoic acid, and on a mixture of nonanoic acid and fluorinated acid cosubstrates. These synthesized PHA have cyanophenoxyhexanoate phenoxyalkanoate, carbon-carbon triple bonds and fluorinated groups at the end of their monomer chain (Fig.3, structures (c), (g), (h), and (i))[18, 23-30]. These functional groups are valuable for further chemical modification. There are many ways to add or replace functional groups on PHA side chains, and consequently, there are many possibilities to modify the physical properties of these PHA. Moreover, current research on these novel PHA is limited by the small amounts available for study. Considering that all the PHA monomers are chiral, they may have some interesting properties that have not yet been discovered.

The  PHA monomer structures described above are dependent on the substrate structures available for the microorganisms. Therefore, it could be possible to design a PHA with the desired functional groups if substrates containing the functional groups are available. There are also many microorganisms that can synthesize PHA with unconventional structures from simple substrates, such as the production of mcl PHA from glucose and gluconate[15, 19]. The synthesis of unconventional PHA from unrelated substrates opens a way to produce low cost PHA. 

3 Synthesis of PHA by Recombinant Bacteria and Transgenic Plants

As early as 1988, the genes for PHB synthesis were reported to be cloned from Alcaligenes eutrophus (now called Ralstonia eutropha) and successfully expressed in Escherichia coli[31, 32]. Since then, many attempts have been made to improve the expression and stabilization of PHB genes in E. coli cells[33-37]. Much attention has also been focused on cloning of different strains and the expression of other PHA genes in various hosts[38-42]. PHA with various monomer structures  including poly-(3-hydroxybutyrate-co-3-polyhydro- xyhexanoate), poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate), mcl PHA, poly-(3-hydroxy-butyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) and poly-(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) have been synthesized by recombinant microorganisms[41-45].

Recombinant DNA techniques may produce new metabolic pathways in the microorganism hosts, thus broadening the usable substrate range, enhancing PHA synthesis capacity and producing a new PHA. It has been reported that recombinant E.coli produced 101 g/L of cell dry weight with 80% PHB content within 39 hours of fermentation in a glucose mineral medium. This is the most efficient PHB production reported so far[45].

Transgenic plants harboring the Ralstonia eutropha PHA synthesis genes have been developed with the aim of ultimately reducing the price of PHA to close to that of starch[46]. The PHB expression level is still very low but as our understanding of plant biochemistry and genetics improves, the PHA expression level will increase. In the near future, PHA production may depend on microbial fermentation.

4 Physical Properties of PHA

The mechanical properties of PHA range from brittle to flexible and elastic, depending on the branched length of the hydroxyalkanoates. The scl PHA such as PHB has high crystallinity and thus PHB is very brittle. However, copolymers consisting of HB and 3-hydroxyvalerate (PHBV) have better mechanical properties than homopolymer PHB[3]. Copolymers consisting of HB and 3-hydroxyhexanoate improve the mechanical properties of PHBV even further[47], making them comparable with conventional plastics such as polypropylene, polystyrene, PET and HDPE (Table 1). 

The  physical properties of mcl PHA are unsuitable for applications due to their low melting points and low glass temperature[48, 49]. They are also elastic but not strong enough for applications. PHA with monomer chain lengths to 12 carbon atoms or more occur as  liquid.

Beside the mechanical properties, various HA monomer structures affect the PHA degradation rates. It has been reported that poly(3-hydroxybutyrate-co-4-hydroxybutyrate) had a much faster degradation rate than homopolymer PHB [50]. However, the lack of sufficient quantity prevents the characterization of other unconventional PHA.

5 PHA Applications

Microbial production of PHA will not be in a position to challenge conventional plastics such as polypropylene and polystyrene that cost only USD 1/kg for a long time[1,4,10,51]. The success of transgenic plants that produce large quantities of PHAs may eventually lower the cost of PHA to a level comparable to conventional plastics. Toward this goal, many studies are still needed to improve PHA gene expression level in economically interesting plants, such as oilseeds and potatoes.

The synthesis of PHA with novel monomer structures may open another interesting area for study. More and more bacterial strains have been isolated from various locations, some with unusual ability to synthesize various PHA.   Some strains have high PHA productivity and produce unusual PHA structures from simple substrates such as glucose and sucrose[52-54]. By using unconventional precursors with functional groups, we may be able to produce a series of PHA with functional groups producing desirable properties, such as enhanced piezoelectricity, nonlinear optical activity, biodegradability and biocompatibility. These high-value-applications will eventually make PHA competitive with other materials possessing similar properties. Recent advances in tissue engineering applications have already shown that PHA promote cell growth on its matrix, so it is a good biomaterial for forming various tissues for medical purposes. The PHA structural diversity has produced different tissue growth promotion effects[55-58].

In addition to applications as plastics, PHA can also represent a potential source of chiral hydroxy acid feedstock for the chemical industry. In contrast to the introduction of new polymers, PHA hydroxy acids and related derivatives can be readily integrated into existing chemical markets[59].The long-term development for PHA is enormous, spanning and benefiting many industries[59]. It will depend on the collaboration of microbiologists, molecular biologists, polymer scientists, material scientists and industry for successes. Many new developments should be expected in the new century.

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