Biopolymers from marine prokaryotes

Biopolymers from marine prokaryotes Ronald M. Weiner Biopolymers from marine prokaryotes, both Bacteria and Archaea, offer a number of novel material properties and commercial marine exopolysaccharides

opportunities.

The characteristics of

and melanins that enhance the survival ability of the

organisms producing them can be exploited for a number of products ranging from emulsifiers

to adhesives. In the prokaryotes,

carbon-storage molecules, but their technological

the polyhydroxyalkanoates

form

application is entirely different,

serving as a potential base material for biodegradable plastics. Marine biopolymers are a significant and undeveloped biological resource.

Prokaryotic biopolymers are an important resource and are finding increasing uses in the biotechnology and biopharmaceutical industries. Capsular, complex extracellular polymeric substances (EPS) alone have multiple industrial applications. For example, Xanthotnonas campestris, a plant pathogen, synthesises xanthan gum (Fig. l), which is found in a number of different foods and has numerous industrial applications’. Similarly, marine biopolymers represent a vast resource of untapped potential, for only a proportion of marine prokaryotes have been cultivated”. This article discusses three general classes of polymers that are synthesised by marine prokaryotes (Table l), and describes their function in nature and their potential and actual commercial applications. The structural, functional and utilitarian diversity of extracellular polymeric substances EPS are usually composed primarily of high molecular weight capsular polysaccharides (CP) and have many important functions in nature, including protecting parasitic organisms from phagocytosis. Their major function in biofilm composition (Fig. 2) and physiology has recently gained much attention, as has the significance of these biofilms3. EPS can function variously as surface adhesins, in protection from predation and desiccation, in ionic interactions, as enzyme ‘stabilisers’, or concentrators, as storage or nutrient reserves and as dispersants (emulsans), to release bacteria from nutrient-spent surfaces”Bs. Function follows structure, and the many roles EPS play in nature are a reflection of their considerable species-specific structural heterogeneity. They range R. M. W&w (n*,[email protected]) is at the Departnwnt ofMicrobiology, L’nivcrsity ofMaryland, College Park, MD 20742, USA. TIBTECH OCTOBER 1997

NOL 15)

Copyright

0 1997,

Elsevier

Science

from simple ol-1-4-linked, unbranched glucose polymers (dextrans) to highly complex, branched and substituted heteropolysaccharides m.ade up of repeating oligosaccharide subunits such as xanthan and colanic acid6. CP can also be substituted with pyruvate, acetate, formate, sulfate, phosphate and other groups. Two identical sugars can bond tea form 11 different disaccharides, whereas two identical amino acids can form only one dipeptide. Additionally, CP contain a wide variety ofsugars’ and, in some cases, several noncarbohydrate side groups. This produces a heterogeneity that far exceeds that of proteins. CP can also be complexed or bonded to vari’ous fatty acids, proteins, hydrocarbons and other polymers of various sizes. Within a species, the length of EPS may be uniform or heterogenous, and their molecular weight ranges from 50 000 to over several million kDa8. The numerous existing and potlzntial applications of EPS are reflections of their many proposed functions in nature4 and their vast array of chemical compositions, bonding structures and architectures. For example, alginic acid (structurally, one of the least complex) is a repeating linear heteropolymer of mannuronic and guluronic acid (Fig. l), with the exact molar ratios varying between species’ and some domains of the polymer having alternating sugars while other domains are homopolymeric. This one type of CP alone has a wide range of applications, based on various properties including its ability to emulsify, retain water, form gels and control ice crystallization. As with other commercially available EPS, even these properties can be modified by subsequent treatments, including, in this case, various salts (ionic alteration) and propylene glycol (covalent alteration). Alginates are ingredients of hundreds of different types of processed food, and are also used in the paper, Ltd. All rights reserved.

0167

- 7799/97/$17.00.

PII: SO167-7799197Jq1099-8

391

marine biotechnology adhesive, textile, ceramics, paint and chemical industries’. Nonmarine bacteria and algae also synthesize alginic acid, which is currently mainly obtained through seaweed harvesting, but it is possible that marine bacteria that synthesize these polymers will soon be exploited leading to more reliable and potentially less expensive product. Rosenberg and co-workers have worked with several EPS with a number of interesting properties. One group ofEPS, isolated from bacteria that colonize fish, has drag-reduction properties; it is speculated that, in nature, these polymers enhance fish mobility, while the surviving fish provide a nutrient-rich substratum’. Another type of EPS are emulsans*O~ll; in nature, emulsans, synthesized most notably by Acinetobucter, are believed to desorb oil-degrading bacteria from ‘spent’ hydrocarbon droplets12. Applications for both of these polymers by industries including petroleum and shipping” are shown in Table 1. An array of promising applications makes use of the ability of many marine bacteria to adhere to various substrates. Marine prokaryotes can attach under pressure, in brine and at temperatures of 4°C or less, and the binding is sufficiently strong to overcome considerable currents3. The adhesive (bonding of dissimilar molecules) properties of some EPS make them promising marine cements’3 and their cohesive (bonding of like molecules) properties, necessary for biofilm accretion, suggest they possess a potential function in coatings. They are also relatively nonimmunogenics, enhancing their potential as bioadhesivesl4. Furthermore, many of the EPS are anionic and can bioconcentrate cations more than 10 000 times’s, An important mechanism for metal scavenging by EPS is the formation of salt bridges between metal ions and the carboxyl groups on acidic polysaccharides, in which polyvalent metal ions bind to two anionic groups on two separate polymer chains. EPS also absorb a variety of organic compounds. These properties make them particularly useful for the bioremedation of heavy metals and metal-containing pollutantsrh. Only a small proportion of the existing EPS have yet been discovered’ and, of these, an even smaller proportion have been characterized. Bacterial melanins While EPS serve as a general example, two additional classes of marine biopolymers are equally promising undeveloped resources. Many of the strains of marine bacteria that have been cultivated express a number of pigments. As with certain EPS, some pigments are also produced by eukaryotic organisms but, in many cases, prokaryotes offer greater potential for commercialization because of the promise of cheaper fermentations and purification. One such class of pigments are the melanins. In nature, although melanins are not essential for growth, they increase the survival capability of the organism in many environments. They protect DNA and other molecules from ultraviolet light, enhance virulence, inhibit biofilm degradation, protect enzymes from protease activity, protect

a CH,OH

6Hm\

1

” coo-

M+y

+M

HO+ Figure 1 Structures of two industrially important capsular extracellular polymeric substances. (a) Xanthan gum, a branched, complex, substituted heteropolymer. (b) Guluronrcacid-mannuronic-acid heteropolymer domain of alginic acid, a linear molecule.

Scanning electron micrograph of a biofilm of a pure culture of Hyphomonas, showing cells attached to the substratum by strands, of extracellular polymeric substances. Bar = 10 Wm. Courtesy of S. Langille.

microorganisms from hydrolytic enzymes and may even act as proton and nutrient sinks in biofilms”. There is considerable heterogeneity among the melanins, although, unlike EPS, they can be classified TIBTECHOCTOBER

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marine biotechnology Table 1. Biopolymers

from marine bacteria with current or potential commercial

application

Types

Polymer

Potential application(s)

Refs

Complex polysaccharides and related extracellular polymeric substances (EPS; normally capsular)

Adhesinsa

Underwater surface coatings, bioadhesives Drilling, ship efficiency Oil cleaning and viscosity reduction Dispersing agent, grinding aid Food, textile, etc. Toxic-metal bioremediation

5,13

Drag reducers Emulsan Surfactant Alginate Metal-binding EPS

9,12

10-12 12 1 16

Pigments

Melanins

Biotechnology,reporter gene, cosmetics,dyes, colorings,sunscreens

17-20

Polyesters

Poly 3-hydroxyalkanoates (e.g. polyhydroxybutyrate)

Biodegradableplastics

23,25,28

aDefined

as prokaryotic

and that may be involved

extracellular in biofilm

polymeric

substances

(EPS) that enable prokaryotes

into several groups based on their synthetic precursors (Table 2). Also unlike EPS, the pigmentation phenotype is rather easily cloned, because generally only a single enzyme is required for melanisationlx. For example, eumelanins originate from the action of tyrosinase’“, while pyomelanins originate from parahydroxyphylpyruvate-hydroxylase activityZo. Although melanins are complex, high molecular weight compounds, their synthesis results from auto-oxidative reactions from active precursors. As such, they are strong reducing and free-radical-capturing agents. Other applications of melanins make use of their chromophoric properties (Table 1). For example, the gene coding for parahydroxyphenylpyruvate hydroxylase, which catalyzes the synthesis of the pyomelanin precursor homogentisic acid (Fig. 3)) may be an excellent reporter gene (indicator of gene expression)

Precursor+

Dihydroxyphenylalanine (DOPA) Eumelanin Phaomelanins DOPAquinoneandglutathioneor cysteine Allomelanins Catechol Pyomelanins Homogentisicacid (alkaptons)

Black Brown, red or yellow Brown Brownor red

(VOL 151

kHp

VH2

H’C-NH2

F=O

LOOH

Pigmentation

aGeneral group of complex polyphenolic heteropolymers. Other (less well documented) classes of melanin also exist”. bThere are a variety of pathways by which tyrosine can be converted to melanin.

TIBTECH OCTOBER 1997

to surfaces,

causing

biofouling,

because: (1) it has a sequence th.at is relatively conserved” and does not form obvious secondary structureiH> and should therefore be efficiently expressed in most species; (2) the end products of its action (homogentisic acid, pyomelanin precursors and pyomelanin) are not toxic; (3) only a single gene need be inserted; (4) the entire sequence is known for the gene from the marine bacterium SIzezva~rcIln wlu~elliar~l~ (shown in batch culture in Fig. 4), leading to easy manipulation; and (5) pyomelanins are obvious, dark red, water-soluble indicators that require no

Table 2. Common types of melanins Melanina

to adhere

accretion.

Tyrosine I

OH

‘COOH

COOH

Para-

Homogentisate

hydroxyphenylpyruvate

Figure3 The precursor to pyomelanin (a low molecular weight melanin) is homogentisate, which is synthesised from phydroxyphenylpyruvate by the action of ghydroxyphenylpyruvate hydroxylase (EC 1.13.11.27). pHydroxyphenylpyruvate is synthesised from tyrosine as part of the tyrosine degradative pathway. When homogentisate is in excess, it can undergo oxidation to form pyomelanin (structure unknown).

/

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marine biotechnology

0-CH-CH2-C

Figure 5 General structure of poly-P-hydroxyalkanoates (PHAs). In poly-13 hydroxybutyrate (PHB), x = 0.

Figure 4 Batch growth of the marine bacterium Shewanella colwelliana in early stationary phase. The brownish-red pigment is pyomelanin and the fluffywhite material is the extracellular polymeric substance. developer. Melanins have a range of chromophoric properties that can also be exploited for sunscreens, dyes and coloring22. Other promising applications take advantage of the fact that melanins sequester different kinds of organic compounds, including fungicides and antibiotics, which may allow them to act as slow-release agents”.

Polyhydroxalkanoates impart environmental plasticity to prokaryotes and environmental compatibility to plastics The final example of marine prokaryotic polymers that have commercial promise are the poly-p-hydroxyalkanoates (PHAs), a class of aliphatic polyesters. This subject has recently been reviewed’3 and, as an important polymer, it is summarized here, emphasising the ecological aspects. In marine prokaryotes, PHAs accumulate intracellularly in large amounts (ranging from 25-80X cellular dry weight) when the organism is in a high-nutrient econiche such as a biofilm, particularly one with a high carbon:nitrogen ratio. As a food reserve, PHAs are metabolised for carbon and energy by prokarotes when they are in low-nutrient econiches such as the mid-ocean. Unlike the earlier examples, complex polysaccharides and pigments, PHAs would be used industrially for a totally different function than they serve in nature; they could be used to form biodegradable, thermoplastic polymers, whereas, in nature, polyesters function as a prokaryotic carbonreserve material. PHAs have the general structure shown in Fig. 5. The lowest molecular weight PHA, poly-p-hydroxybutyrate (PHB), is the most common in nature. However, their molecular weights can be up to hvo milion, which corresponds to approximately 20 000 monomers per polymer molecule. Many prokaryotes, for example halobacteria’s and hyphomicrobia26,

synthesise PHAs and, when carbon and energy are required, the PHA is normally depolymerized to D(-) hydroxybutyric acid and then metabolised to acetoacetate and acetoacety-CoA. The usefulness of the prokaryotic PHAs comes from their thermoplasticity, a property found in numerous plastics including polystyrene, polycarbonate and polypropylene. The significant difference is that prokaryotic PHAs form the base material of biodegradable plastics 25; for example, PHB has a similar structure to polypropylene, which is used in over 200 products, including pipes, packaging malterials and domestic appliances. Biodegradability is enhanced because, unlike (synthetic) polypropylene, (prokaryotic) PHB does not float, but sinks to the sediment, where it can be degraded in approximately one month, rather than the year it would have taken had it remained on the surface, all other conditions being equa127. The properties of PHAs, like those of the complex polysaccharides and pigments described above, can be altered and enhanced by manipulating or modifying their chemical composition. For example, homopolymers of PHB are brittle, whereas copolymers of PHB and 3-hydroxyvalerate have lower melting temperatures, resulting in increased flexibility and resiliency’s. Copolymers of various proportions of each building block can be engineered by manipulating the carbon sources supplied during growth’s, Until an economical high-yielding marine-growth medium is formulated, the development of geneticengineering methods for PHA synthesis is the most promising method of production. In Alcdl@tzes etrtr@us, for example, there are three genes for the synthesis of PHB that have been successfully cloned”. The cloning vehicle can be chosen to produce high yields in existing growth media. In either case, for the foreseeable future, PHA will be produced by prokar!iotic fermentations’s, using either natural or engineered organisms. It might be quite some time, if ever, befo:re transgenic plant technology is possible and cost effective. While prokaryotic polymers remain more costly to produce than those from other sources, the key to their future (their minimal impact on environmental deterioration) lies in two facts - they are biorenewable and many are biodegradable and/or nontoxic. For example, in 1982, ICI began to market PHA-derived TIBTECH OCTOBER 1997 (VOL 15)

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marine biotechnology

“and the marine biopolymers plastics under the trade name Biopol. While Biopol is still more than twice as costly to produce as other plastics, it remains commercially important because it is biodegradable and so, when environmental remediation is also considered in the productivity equation, Biopol becomes cost competitive in an environmentally conscious society. Isolations of novel marine prokaryotes are becoming commonplace. There have been innovative advances in fermentation technology and analytical chemistry, leading to better product characterisations. With these developments, many more marine prokaryotic biopolymers will find their way to the marketplace. Acknowledgments Special thanks go to R. Colwell and E. Rosenberg, both of whom made substantial contributions to the development of marine biotechnology, and provided valuable insights to this paper. A grant from the Maryland Industrial Partnerships and Oceanix Corp. supported RW. References 1 Sandford, P. A. (1984) in Biofechnolo~y oj Marine Polysac~haridr~ (Colwell, R. R., Pariser, E. R. and Sinksey, A. J., rds), pp. 454-516, McGraw-Hill 2 Hugenholtz, P. and Pace, N. R. (1996) Trends Biotechnoi. 14, 19&197 3 Potera, C. (1996) Science 273, 1795-1797 4 Dudman, W. F. (1977) 1x1Swj& Carbuhydratex ojfhe Prookaryotic Cell (Sutherland, I. W., ed.), pp. 357-414, Acadermc Press 5 Werner, R., Langille, S. and Qumtero, E. (1995)j. Itid. Muobiol. 15, 339-346 6 Christensen, B., KJosbakken, J. and Smtdsrod (1985) Appl. Environ. ,I&obiol. 50, 837-845 TIBTECH OCTOBER 1997 (VOL 151

are particularly

good this year!”

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