PHA polímero

PHA polímero

(Parte 1 de 7)

Perspectives for Biotechnological Production and Utilization of Biopolymers: Metabolic Engineering of Polyhydroxyalkanoate Biosynthesis Pathways as a Successful Example

Alexander Steinbuchel Institut fur Mikrobiologie, Westfalische Wilhelms-Universitat Munster, Corrensstraße 3, D-48149 Munster, Germany

I Occurrence, Structures, Biosynthesis Principles and Functions of Biopolymers

Living matter is able to synthesize a wide range of different polymers, and in most organisms these biopolymers contribute the major fraction of cellular dry matter. The functions of biopolymers are, in most cases, essential for the cells and are as manifold as their structures.[1]

Chemical Classes of Biopolymers

Organisms are able to synthesize an overwhelming variety of polymers which can be distinguished into eight major classes according to their chemical structure: (i) nucleic acids, (i) polyamides such as proteins and poly- (amino acids), (i) polysaccharides, (iv) organic polyoxoesters such as poly(hydroxyalkanoic acids), poly(malic acid) and cutin, (v) polythioesters, which were only reported recently,[2] (vi) inorganic polyesters with polyphosphate as the only example, (vii) polyisoprenoides such as natural rubber or Gutta Percha and (viii) polyphenols such as lignin or humic acids (Table 1).

Functions of Biopolymers

These biopolymers fulfil a range of quite different essential functions for the organisms such as conservation and

Review: This article provides an overview of biopolymers, classed according to their chemical structures, function and occurrence, the principles of biosynthesis and metabolism in organisms. It will then focus on polyhydroxyalkanoates (PHA) for which technical applications in several areas are currently considered. PHAs represent a complex class of bacterial polyesters consisting of various hydroxyalkanoic acids that are synthesized by bacteria as storage compounds for energy and carbon if a carbon source is present in excess. Poly(3-hydroxybutyrate), poly(3HB), is just one example. Most other PHAs are only synthesized if pathways exist which mediate between central intermediates of the metabolism or special precursor substrates on one side and coenzyme A thioesters of hydroxyalkanoic acids, which are the substrates of the PHA synthase catalyzing the polymerization, on the other side. During the last decade, basic and applied research have revealed much knowledge about the biochemical and molecular basis of the enzymatic processes for the synthesis of PHAs in microorganisms. The combination of detailed physiological studies, utilization of the overwhelming information provided by the numerous genome sequencing projects, application of recombinant DNA technology, engineering of metabolic pathways or enzymes and molecular breeding techniques applied to plants have provided new perspectives to produce these technically interesting biopolymers by novel or significantly improved biotechnological processes or by agriculture. Some examples for successful in vivo and in vitro engineering of pathways suitable for the synthesis and biotechnological production of PHAs consisting of medium-chain-length 3-hydroxyalkanoic acids and shortchain-length hydroxyalkanoic acids will be provided.

Integration of an in vitro engineered poly(3HB) biosynthesis pathway into the metabolism of E. coli.

2 A. Steinbuchel expression of genetic information, catalysis of reactions, storage of carbon, energy or other nurients, defending and protecting against the attack of other cells, hazadous environmental factors, sensing of biotic and abiotic factors, communication with the environment and other organisms, mediation of the adhesion to surfaces of other organisms or of non-living matter and many more. Alternatively, they may be structural components of cells, tissues and entire organisms. To fulfil these different functions, biopolymers must exhibit some unique properties. Microorganisms can synthesize biopolymers belonging to the classes (i) to (vi), and among them are numerous polymers which are used by industry for technical applications in medicine, pharmacy and agriculture, as packaging materials and in many other areas. Biotechnological production of these polymers is, at present, mostly achieved by the fermentation of microorganisms in stirred-tank bioreactors, and the biopolymers can be obtained as extracellular or intracellular compounds. Alternatively, biopolymers can be also produced by enzymatic in vitro processes. Biopolymers belonging to the classes (vii) and (viii) are mainly synthesized by eukaryotic organisms and most abundantly by plants.

Principles of Biopolymer Synthesis: Location of Biosynthesis

All biopolymers are synthesized by enzymatic processes in the cytoplasm, in the various compartments or organelles of cells, at the cytoplasmic membrane or at cell wall components, at the surface of cells or even extracellularly. Synthesis of a biopolymer may be initiated in one part of a cell and may be continued in another part as it occurs, for example, during the synthesis of complex cell wall constituents in bacteria. There are also numerous examples of the transport of polymers from one compartment of a cell to another as it may be required, for example, for some proteins in the mitochondria, and chloroplasts in eukaryotic organisms. Polymers can be also excreted from cells into the environment. This occurs, for

Table 1. Seven classes of biopolymers: characteristics of their biosynthesis and occurrence.

Class Template- Substrate of the polymerase Synthesis in dependent synthesis Prokaryotes Eukaryotes

1. Polynucleotides Nucleic acids yes dNTPs, NTPs yes yes 2. Polyamides Proteins yes Aminoacyl-tRNAs yes yes

Poly(amino acids) no Amino acids yes yes 3. Polysaccharides no Sugar-NDP, Sucrose yes yes 4. Polyoxoesters no Hydroxyacyl coenzyme A yes (no) 5. Polythioesters no Mercaptoacyl coenzyme A yes no 6. Polyphosphate no ATP yes yes 7. Polyisoprenoids no Isopentenylpyrophosphate no plants, some fungi 8. Polyphenols e.g. Lignin no Radicalic intermediates no only plants

Prof. Dr. Alexander Steinbuchel was born in Luneburg (FRG) 47 years ago. His interest in microbiology began with undergraduate studies at the University of Gottingen and was extended through his research work in the field of enzyme fermentation undertaken for his Diploma and PhD theses with Prof. Schlegel at the same university. After a year at the Rockerfeller University (New York) in the department of Prof. Christian DeDuve, he returned to the University of Gotingen where he completed his habilitation in 1991. He spent one month as a Visiting Professor at the University of Buenos Aires and since September 1994 has held the Chair of Full Professor of Microbiology and has been the Director of the Institut fur Mikrobiologie at the Westfalische Wilhelms-Universitat Munster. His current areas of interest are the physiology, biochemistry and genetics of (i) metabolism and biotechnological production of polyhydroxyalkanotes, (i) biosynthesis and biotechnological production of polyamides, (ii) degradation of natural and chemosynthetic polymers, (iv) microbial degradation of actoin, (v) regulation of fermentative metabolism in aerobic bacteria and (vi) microbial transformation of flavour compunds. A highlight of his career is the award of the “Philip Morris Forschungspreis” received with H. G. Schlegel and G. Gottschalk for the production of biodegradable thermoplastic polyesters from renewable resources. He has over 200 publications to his name and is an active member of the editorial boards of many journals including Macromoecular Bioscience. Prof. Dr. Alexander Steinbuchel is married and has 3 children.

Perspectives for Biotechnological Production and Utilization of Biopolymers ... 3

example, in enzyme proteins which hydrolyze polymeric nutrients or lipids. Furthermore, polymers such as plasmids or other parts of the genomes can be taken up by cells in processes referred to as transformation or conjugation.[1,3]

Principles of Biopolymer Synthesis: Templatedependent and -independent Processes

Biopolymers are either synthesized by template-dependant or template-independant enzymatic processes. This difference has significant consequences for the structure and the molecular weight of the polymers. Nucleic acids are synthesized with desoxyribonucleic acid (DNA) or ribonucleic acid (RNA) as a template, whereas messenger RNA (mRNA) is the template for the synthesis of proteins by ribosomes (Table 1). Nucleic acids and proteins exhibit a complex primary structure in which the constituents are, from a purely chemical point of view, randomly distributed. A template guarantees that the complex primary structure of these two classes of polymers is highly conserved. Another consequence of the templatedependant biosynthesis of polymers is that the resulting polymers are monodisperse, i.e., consist of individual molecules all possessing exactly the same molecular weight. In contrast, poly(amino acids) and members of the classes (i) to (vi), which are synthesized by template-independant enzymatic processes (Table 1), are polydisperse, i.e., the individual molecules of a particular biopolymer species do not exhibit a uniform molecular weight. If the molecular weight of the latter is described, usually the weight average molecular weight and the molecular weight distribution indicated by the polydispersity index are analyzed, in the same way as for polymers obtained from synthetic processes.[4]

Principles of Polymer Biosynthesis: Substrates of the Polymerizing Enzymes

Most biopolymers are not simply synthesized by the direct polymerization of their building blocks. For example, glycogen is not synthesized by the polymerization of glucose, but from ADP-glucose or UDP-glucose.[5] Table 1 lists in a general way those intermediates of the metabolism that are used by the polymerizing enzyme systems as substrates. If no activated or energy-rich compound is used for the polymerization reaction, the reaction may be driven by the hydrolysis of ATP as, for example, during biosynthesis of poly(c-d-glutamate)[6] or another energy-rich cosubstrate. Well known exceptions from these two rules are the synthesis of, for example, dextran and some other glucans or fructans.[7] A special situation occurs during the synthesis of lignin which relies on the enzymatic formation of radicals derived from phenylpropane units and spontaneous chemical reaction.[8]

Biopolymers and Renewable Resources

In addition, biopolymers can be obtained from agriculture or from biotechnological processes and are therefore, in principle, available from renewable resources. Figure 1 provides an overview on the major products which are available from plantations of crops and trees (i.e., from agriculture and forestry) and which are used for non-food applications. With the exception of triacylglycerols and sucrose, these products are mainly polymers such as starch, cellulose, lignin and natural rubber. By far the most important producers are plants. Autotrophic microorganisms may be also candidates for the synthesis of some of these biopolymers; however, as yet, only weak perspectives for a biotechnological production of biopo-

Figure 1. Major renewable carbon sources available from plants and autotrophic microorganisms.

4 A. Steinbuchel lymers have been outlined, and only for polyhydroxyalkanoates employing chemolithoautotrophic[9] and photoautotrophic[10] microorganisms. In contrast, heterotrophic microorganisms are considered as suitable producers of several biopolymers such as PHAs, polysaccharides and polyamides. Many biopolymers possess rather complex chemical structures and compositions and are therefore not available from chemical synthesis. Furthermore, biopolymers, like almost all products of living matter, are generally biodegradable, whereas this is not a general feature of synthetic polymers.[1,12] These few aspects indicate reasons for the growing interest of industry to produce and use biopolymers for a steadily increasing number of applications.[13] Only few biopolymers have been commercialized so far; this is mainly due to the production costs, which are often much higher than for synthetic polymers that have been established in the market during the last decades. However, this situation may change for some biopolymers in the future, when biotechnological production processes have been further optimized and if our knowledge of the material properties and processing of biopolymers has increased.

Isolation and Production of Biopolymers

There are different ways to produce biopolymers in order to make them available for interesting technical applications: (i) Many biopolymers occur abundantly in nature and are isolated from plants and algae which grow in natural environments or are cultivated on plantations. Cellulose and starch are isolated from several agricultural crops and trees such as Zea maize or Pinus silvestris, respectively. Natural rubber is isolated from plantantations of the rubber tree Hevea brasiliensis, and agar and alginates are isolated from red algae belonging to the genus Gelidium[14] or from various brown algae also referred to as seaweeds,[15] respectively. (i) Few biopolymers are isolated from extremely scarce natural sources. An example of such an exception is hyaluronic acid which is extracted from the umbilical cords of new born children.[16] (i) In vitro synthesis of biopolymers with isolated enzymes in cell-free systems offers another possibility to produce biopolymers. One example is the application of the heat-stable DNA polymerases in the polymerase chain reaction (PCR) to produce monodisperse defined DNA molecules.[17] Another example is dextran, which can be produced on a technical scale with isolated dextran sucrase.[18] (iv) Fermentative production of biopolymers is used by industry to obtain, for example, polysaccharides such as xanthan and dextran by employing the bacteria Xanthomonas campestris[19] or Leuconostoc mesenteroides,[7] respectively. Microbial cellulose obtained by fermentation of Acetobacter xylinum seems to offer some advantages for medical applications over cellulose which has been isolated from plants.[5] The homopolyester poly(3-hydroxybutyrate), poly(3HB), and the copolyester poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate)], poly(3HB-co-3HV), which were available under the trade name “Biopol” from ZENECA and Monsanto, have been also produced on an industrial scale by employing Ralstonia eutropha.[20] The aforementioned examples are not based on genetically modified bacteria; however, genetically engineered bacteria can also be employed for the production of biopolymers. (v) Finally, there are many efforts to generate transgenic plants for the production of certain biopolymers. Examples are plants producing Biopol[21] or amylose and amylopectin.[2]

I Impacts of Advanced Technologies and Methodologies on Biopolymers

There are many reasons for scientists from academia and industry to be interested in biopolymers. Firstly, during the investigation of biological aspects of biopolymers and their metabolism scientists will still find many “white spots” since our knowledge about the physiology, biochemistry and molecular genetics of biopolymers is often scarce. Secondly, many biopolymers have unique properties, and practically all of them are biodegradable in contrast to most synthetic polymers. Thirdly, since chemical synthesis is neither possible nor economically feasible, even if the structure of a biopolymer seems to be not too complex, organisms are often the only source for these polymers which can therefore be obtained at lower costs or higher purity. They are either isolated directly from higher organisms as they occur in nature or on plantations of agricultural crops or trees or they are biotechnologically produced by the fermentation of microorganisms. Therefore, they are directly or indirectly available from

CO2 or renewable resources. During the last decade basic and applied research have revealed much knowledge on the biochemical and molecular basis of the enzymatic processes for polymer synthesis in microorganisms. The combination of detailed physiological studies, utilization of the overwhelming information provided by the numerous genome sequencing projects, application of recombinant DNA technology, engineering of metabolic pathways or enzymes, and molecular breeding techniques applied to plants, provided new perspectives to produce technically interesting biopolymers by novel or significantly improved biotechnological processes or by agriculture. Geneticists and molecular biologists have developed many powerful methods for in vitro and in vivo modification of DNA.[23] In addition, efficient methods are available for the transfer of DNA and expression of heterologous genes which are applicable to many organisms. Metabolic engineering has become an important and powerful approach in biotechnology[24] (Table 2). Successful metabolic engineering

Perspectives for Biotechnological Production and Utilization of Biopolymers ... 5 requires, however, a detailed knowledge of the physiology and metabolism of the respective organism to which these technologies are applied. It also requires knowledge and experience to utilize the large diversity of microorganisms for example as sources for useful genes.[25,26] Scientists knowing only E. coli or being only perfect in DNA sequencing will have problems, as will scientists who have never worked on a larger scale than that of an agar plate or a tiny reaction tube.

I Intracellular versus Extracellular Production of Biopolymers

The biotechnological production of biopolymers may occur intracellularly or extracellularly. This causes several severe consequences regarding the limitations of the production and downstream processes to obtain the biopolymers in a purified state (Figure 2).

PHAs,[27] cyanophycin,[28] glycogen,[29] starch[30] and polyphosphate[31] are examples of biopolymers which are accumulated in the cytoplasm of cells. The availibility of space in the cytoplasm therefore limits the amount of polymer that can be produced by a cell. This is particularly relevant for fermentative production processes mostly employing microorganisms. Therefore, the yield per volume is limited/determined by the cell density and the fraction of the biopolymer in the biomass. Another general consequence is that more or less tedious processes must follow the production of the biomass containing the biopolymer to disintegrate the cells or tissues and to release the biopolymer from the cells. Furthermore, other cell constituents will be released concommitantly with the biopolymer and must then be separated.

Poly(c-d-glutamate)[32] and many polysaccharides, such as alginates,[15] dextran,[7] xanthan,[19] and microbial cellulose[5] are examples of biopolymers which occur outside the cells, either as a result of extracellular synthesis or of excretion by the cells. For these biopolymers, the volume of the cytoplasm is not a limiting factor, and, in principle, the entire volume of the bioreactor (instead of only that of the cytoplasm) would be available to deposit the desired biopolymer. Furthermore, breakage of cells or tissues is not required and separation of the biopolymer from the other biomass is not very complex. However, biotechnological processes can merely take advantage of these features since the presence of these mostly watersoluble biopolymers in the medium usually causes a high viscosity in the medium, resulting in rheological problems during the fermentation process. Unfortunately, hydrophobic water-insoluble biopolymers of biotechnological interest that occur extracellularly are not known. Therefore, in practice, the amount of biopolymer produced per volume by extracellular processes is usually lower than can be obtained by intracellular processes.

Other strategies and the use of cell-free production processes, may take advantage of the features of extracellular processes. One strategy is to apply in vitro synthesis of biopolymers employing isolated enzymes. Another strategy is to produce the constituents of polymers as monomers by fermentative processes and to polymerize these components subsequently by solely chemical processes. Both these strategies have already entered reality and many different examples of scale have been demonstrated (i.e., not only at the laboratory scale but also at the technical scale). Polylactide acid, for example, will be produced on a large scale by such a combined biotechnological and chemical approach.[3] The third and most difficult and pretentious strategy would be to convert an intracellular process into an extracellular process by metabolic and genetic engineering of the cells or organisms. This could be done by, for example, extending the capability of a cell to excrete a polymer which is synthesized intracellularly. However, to the best knowledge of the author, this has not been demonstrated, yet.

IV Occurrence of Natural Polyesters

Three different types of naturally occurring organic polyesters and one inorganic polyester are known (Figure 3). These are polyoxyesters occurring in the prokaryotic bacteria and archaea, polymalic acid occurring in

Figure 2. Examples of biopolymers which are produced intracellularly and extracellularly and a demonstration of the “volume” problem.

Table 2. Tools for successful and powerful metabolic engineering.

0 A large box of sophisticated in vitro and in vivo molecular methods for modification of DNA and for transfer of recombinant DNA into other organisms 0 Detailed knowledge on physiology and metabolism 0 Diversity of microorganisms 0 Genome sequencing projects provide an overwhelming amount of data

6 A. Steinbuchel

eukaryotic microorganisms, and cutin and suberin occurring in plants. In addition, the inorganic polyester polyphosphate occurs in organisms of all the kingdoms. As far as it is known, all these types of polyesters are synthesized by different mechanisms and are catalyzed by enzymes exhibiting quite different characteristics.

Bacterial Polyoxyesters

An overwhelming number of different polyhydroxyalkanoates (PHAs), comprising approximately 150 different hydroxyalkanoic acids as constituents, has been isolated from bacteria during the last 20 years.[34] Accumulation of PHAs in the bacterial cell usually occurs if a carbon source is provided in excess, and if at least one other nutrient, which is essential for growth, has been depleted, i.e., if growth is imbalanced.[27,35] These water insoluble polyesters accumulate in the cytoplasm and are deposited as cytoplasmic inclusions which are referred to as PHA granules.[36] They serve as storage compounds for energy and carbon. PHAs are synthesized by diverting either central intermediates of the carbon metabolism or derivatives from precursor substrates, which are provided as carbon source for the growth of the bacteria, to hydroxyacyl-CoA thioesters.[37] The thioesters are then polymerized by PHA synthases[38,39] that are bound to the surface of PHA granules together with other proteins.[36,40,41]

Polymalic Acid

This is the only water-soluble polyester occurring in living matter.[42] This anionic homopolyester is synthesized by Physarum polycephalum[43,4] and a few lower eukaryotic microorganism such as Penicillium cyclopium[45] and Aureobasidium pullulans.[46,47] So far, no bacteria have been identified which synthesize polymalic acid. The biosynthesis of this polyester is, as yet, only poorly understood, and polymerization seems to occur by a mechanism different from that of the bacterial polyoxoesters.[48] This biopolyester is mentioned here only for completness and will not be further considered in this review.

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