Tools and Applications of Biochemical Engineering Science

of the stirring rate. This also implies that when the process is scaled-up to a large scale, keeping the shear stress constant, the overall infection rate should be the same independent of the higher turbulence regime at the larger scale [98].

From the Dee and Shuler model the virus depletion rate function can be defined as [106]:

Where C is the cell concentration (cells/ml),Vex is concentration of free virus in suspension (pfu/ml) and ka is the attachment constant (ml/cells h–1).

To know the binding kinetics is also important in MOI-TOI specification, in particular at low MOI’s, for different cell lines. T. ni cells like HighFive‘ have a constant of attachment almost 10-fold higher than S. frugiperda cells such as Sf9 [60].

Considering equal time steps (Dt) Dee and Shuler propose that the amount of virus bound during the first time step could be approximated by:

Where C1 is the cell concentration after time step 1.

In the Licari and Bailey Model [102] and also in the latest Hu and Bentley

a-value has been proposed to be dependent on the physical system and a value of a = 0.04 was proposed for static systems [102]. For agitated systems suspension cultures Hu and Bentley proposed a value of a = 0.08 because they state that agitation systems enable higher efficiency of contact between viruses and cells [105]. This is not absolutely true, at least the true reason is not the higher mixing level but the fact that in static cultures, less cell surface is exposed to the virus, since to the cells are attached to a surface. This gives an overall constant of attachment 3-4 fold lower than in suspension systems [61].

Licari and Bailey used the Poisson distribution in the following form:

p(t, j) is the probability at time t that a cell could be infected by j viruses, t is the time (h), j is the number of viruses infecting one cell, a is the empirical factor and MOI(t) is the Multiplicity of Infection at time t.

The sum of all possible combinations of the number of viruses that could be infecting one cell is given by:

expression occurs in a transient mode, the models in general fit the data with good correlation.

The Hu and Bentley model is the only one that tries to describe VLP production and assembling in baculovirus infected insect cells [105]. Nevertheless, regarding VLP assembly, the formalism presented is completely theoretical and based on the assembly pathway of icosahedral viruses. From a process development point of view, this model does not generate enough output to make it applicable to bioreaction operational parameters definition. However, it can be used as the basis for a more structured approach to the VLP assembling process in baculovirus infected insect cells.

The application of mathematical modelling to baculovirus infection and vi- rus-like particle production was also successfully done to Parvovirus B19 viruslike particle production with two different baculovirus at low MOI’s [18]. But in this model the same concepts proposed in the Licari and Bailey Model was applied, i.e. baculovirus infection follows Poisson distribution with mean and variance equal to a.MOI, but with co-infection with two single-vectors, each one encoding a specific viral protein.

However, as important as the Hu and Bentley Model is the stepwise approach to process optimisation that Hu and Bentley have reported [33]. The focus on quantitative analysis of protease degradation of the product over time, along with the similar approach followed by Cruz et al. [25], also indicate new directions to follow in mathematical modelling regarding product expression optimisation.

Mathematically modelling of VLP production by baculovirus infected insect cells is not a closed chapter of research, and probably never will be, at least until complete structured models have been developed, taking into account intracellular pools.

James Bailey has said, talking about the inextricable coupling between a model and its intended application (citing Casti) that “Basically the point of making models is to be able to bring a measure of order to our experience and observations, as well as to make specific predictions about certain aspects of the world we experience” [108]. So models are not an end in themselves but should be looked more as tools to think and calculate logically about what components and interactions are important in a complex system. Mathematical models can also be justifiable to minimize the experimental effort, predict system behaviour and identify new research avenues [10].

6 Conclusion

The production of VLPs with baculovirus infected insect cells is entering the phase of industrial application. Due to the complexity of the system a multitude of vector strategies have been developed, ranging from single to fifth-level baculovirus vectors, along with combinations between them.

Bioreaction optimisation involves assessment of good oxygen transfer conditions and evaluation of shear stress influence on cell growth and productivity.

Scalability of the systems is a must and the use of low MOI strategies is obligatory if the goal is the industrial application. Complex feeding strategies must be avoided since they impose high risks, and costs, to the overall process.

Better mathematical models providing a better understanding of the basic events are still needed, incorporating the attachment steps, the intracellular events, and product expression, assembling and degradation.

Acknowledgement. The authors acknowledge and appreciate the financial support received from the European Commission (BIO4-CT98-0215) and from Fundação para a Ciência e Tecnologia – Portugal (PRAXIS XXI/BD/16136/98).

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Integrated Approach To Explore the Potential of Marine Microorganisms for the Production of Bioactive Metabolites

Irene Wagner-Döbler1, Winfried Beil2, Siegmund Lang3,*, Marinus Meiners4,

Hartmut Laatsch5

1Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, 38124 Braunschweig, Germany

E-mail: iwd@gbf.de

2Institut für Allgemeine Pharmakologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, 30625 Hannover, Germany

E-mail: beil.winfried@mh.hannover.de

3Institut für Biochemie und Biotechnologie, TU Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany

E-mail: s.lang@tu-bs.de

4Fachbereich Naturwissenschaftliche Technik, Fachhochschule Ostfriesland, Constantiaplatz 4, 26726 Emden, Germany

E-mail: meiners@nt-newton.fho-emden.de

5Institut für Organische Chemie, Universität Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany

E-mail: hlaatsc@gwdg.de

Dedicated to Prof. Dr. Wolf-Dieter Deckwer on the occasion of his 60th birthday

During the last 10 years marine organisms have provided a large number of new natural products. Interesting compounds have mainly been derived from macroorganisms such as sponges, ascidians, corals and bryozoans. The number of secondary metabolites from marine microorganisms is smaller, but rapidly increasing. Because of the enormous difficulties involved in harvesting products from marine animals, and the fact that some of the bioactive compounds are produced by associated bacteria, the advantages of sustainable production of bioactive metabolites by bacteria or fungi, under the protection of natural resources, seem to be very attractive for the future. This review describes current progress in the isolation and identification of novel marine microorganisms, the discovery of new secondary metabolites, the biotechnological approaches to overproduce them, as well as the evaluation and characterization of their bioactivity.

Keywords. New bacteria from the North Sea, Secondary metabolites, Bioactivity, Biotechnological production, Structure elucidation, Phylogenetic screening

1Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

1.1Marine Bacteria as Sources of New Bioactive Compounds . . . . . . 208

* To whom all correspondence should be addressed.

Advances in Biochemical Engineering/

Biotechnology, Vol. 74

Managing Editor: Th. Scheper

© Springer-Verlag Berlin Heidelberg 2002

2Molecular Tools for Phylogenetic Screening of the Marine Microbial Diversity and First Results on an Indigenous Group of Marine

5Biotechnological Studies To Overproduce Metabolites

1 Introduction

1.1

Marine Bacteria as Sources of New Bioactive Compounds

The search for bioactive compounds is presently reaching a new dimension – with such diverse approaches as genomics, proteomics, bioinformatics, combinatorial biosynthesis, combinatorial chemistry, targeted drug development, directed evolution of key enzymes, phage display libraries, automation and high throughput screening [1–4]. What role can traditional natural product screening play in today’s drug development approaches [5], and what in particular

Fig. 1. Total number of metabolites from marine microorganisms (1966–1999)

can be expected from marine microorganisms [6, 7] as sources of novel compounds?

Marine organisms have provided a seemingly endless parade of novel structures. New carbon skeletons were described with a frequency that exceeded all expectations. Several structural features are uniquely or predominantly marine (reviewed by Faulkner since 1986 [8–10]). Thus, ample raw material was provided for combinatorial chemistry, synthetic chemistry, and biocatalysis [11]. The most striking change in the direction of marine natural product chemistry since 1993 was reflected in the sudden increase in reports of new metabolites from marine microorganisms (Fig. 1): Whereas less than ten compounds had been known 20 years ago [12], the number of structures has exponentially grown since then. Although a total number of around 400 compounds is still small in comparison with more than 15,000 structures from terrestrial microorganisms, new compounds are presently almost exclusively found in marine organisms (W Fenical, personal communication).

A number of invertebrate phyla is restricted to the marine environment, with no or few representatives in aquatic and terrestrial habitats, e.g., the Ascidia, Porifera, Coelenterata and Bryozoa. Consequently, from these organisms, a whole range of new chemical structures has been discovered that are not found in terrestrial organisms. However, in many cases, it is not yet clear whether the bioactive compound is produced by the invertebrate, by endosymbiontic or epiobiotic bacteria, or in a cooperative way by both. There is increasing evidence for an important role of the bacterial endosymbionts in the bryostatinproducing bryozoon Bugula neritina [13, 14] or for metabolite production in demospongiae [15].

1.2

The Phylogenetic Tree and the Production of Bioactive Compounds

What are marine bacteria? Defining them as bacteria with an absolute requirement for sodium chloride is not a practical solution, because many marine isolates may tolerate quite a wide range of salinities, prompting speculation that they are in fact terrestrial organisms that have been swamped into the oceans from rivers, estuaries and sewage outfalls. Pragmatically, marine microorganisms are therefore defined as bacteria that have been isolated from marine sources on marine media [16].

However, one has to bear in mind that the microbial ecology of marine habitats has been revolutionized by cultivation-independent analyses based on 16S rRNA. It is now well documented that only a fraction of the marine microbial diversity has been cultivated, presumably far less than 1% [17], no more than the “tip of the iceberg” [18]. Clone libraries of marine bacterioplankton 16S rRNA genes are dominated by a few phylotypes that have not been cultivated to date, and which are distributed globally [19, 20]. It can therefore be concluded that the “true” marine microorganisms are in most cases presently not known.

Within the operationally defined “marine bacteria”, i.e., bacteria isolated from marine samples on marine media, bioactive compounds have been reported from Pseudoalteromonas, Cytophaga, Alteromonas, Micrococcus, Bacillus, Acinetobacter, Agrobacterium and Pseudomonas or from unidentified bacteria (Fig. 2).

Cultivated marine bacteria are scattered throughout the phylogenetic tree of the domain Bacteria. However, at lower phylogenetic levels, clusters of marine bacteria have been found which are distinct from those of terrestrial origin. One example is the so-called a3-subgroup of the a-Proteobacteria subclass of the division Proteobacteria, the Roseobacter clade [20]. A marine group of Actinobacteria [21] has been described, which has, to date, however not been cultivated.

Presently, there are three microbial phylogenetic hot spots known for the production of secondary metabolites:

1.The Streptomycetes, a group of filamentous Gram-positive bacteria (Actinomycetes) that are the work horses of natural product isolation [22];

2.the Myxobacteria, motile bacteria with a complex life cycle which form a distinct cluster within the d-subclass of the Proteobacteria and have shown to be a rich source of novel structures and biological activities [23, 24];

3.the Cyanobacteria, the former bluegreen algae, photosynthetic bacteria which are distributed globally and produce extremely potent toxins (e.g., [25–27]).

In addition, the antibiotics and other bacteriocins were originally detected in lactic acid bacteria, but were later also found in other Gram-positive microorganisms [28]. Lactic acid bacteria are a group of non-spore-forming, anaerobic fermentative bacteria within the Gram-positives with low GC content.