Genomics Based Approaches in Fermentation Technologies
ID: 3932

Abstract: A.J.M Driessen

A.J.M. Driessen 1, I.J. van der Klei 1, J.D. Sutherland 2, R.A.L. Bovenberg 1,3. 1 University of Groningen, Netherlands 2 University of Manchester, U.K., and 3 DSM Anti-Infectives, Delft, Netherlands

We would like to report on a project that aims for breakthrough bioprocess technology for the production of 7-amino-cephalosporanic acid (7-ACA) derived semi-synthetic cephalosporins, by novel integration of fermentation of pathway engineered fungi with enzymatic and organic chemical synthesis steps. This diverse class of compounds represents a steady economical value of over $4 bio/year. Typically, current synthesis is based upon fermentation of Cephalosporin C, isolation and chemical conversion into 7-ACA and subsequent multi-step chemical synthesis into the various final products. The isolation and chemical conversions are notoriously complex and noxious process steps.

Therefore we engineered the expression of different heterologous enzymes in the filamentous fungus Penicillium chrysogenum to convert the natural penicillin pathway into a new pathway leading to an advanced cephalosporin compound, adipoyl-7-carbamoylcephalosporanic acid (adACCA). 1 This product is a convenient stable intermediate that can easily be isolated and integrated in organic-chemical schemes for the further conversion into semi-synthetic antibiotics. The adipoyl side chain is an attractive protective group, which can be removed by enzymatic means using mild process conditions. Bio catalytic production of adACCA would provide a real breakthrough in the field of manufacturing of advanced cephalosporin antibiotics.

We will report on Penicillium chrysogenum mediated adACCA biosynthesis, on membrane transporters important for product secretion [1], the enzyme localisation for optimal pathway performance in compartmentalised fungal mycelium and further recent insights obtained by a genomic analysis of P. chrysogenum [2].

References:

1. Nijland, J.G., Kovalchuk, A., van den Berg, M.A., Bovenberg, R.A.L., Driessen, A.J.M. (2008) Expression of the Transporter Encoded by the cefT gene of Acremonium chrysogenum Increases Cephalosporin Production in Penicillium chrysogenum. Fungal Genetics Biol. 45: 1415-1421.

2.van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.-M., Driessen, A.J.M., Estrada, C.G., Fedorova, N.D., Harris, D., Heijne, W., Joardar, V., Kiel, A.K.W., Kovalchuk, A., Martin, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der Klei, I.J., van Peij, N.N.M.E., Veenhuis, M., von Döhren, H., Wagner, C., Wortman, J., Bovenberg, R.A.L. (2008) Genome Sequencing and Analysis of the Filamentous Fungus Penicillium chrysogenum Wisconsin54-1255. Nature Biotechnol. 26: 1161-1168.



Jeroen Hugenholtz

In industrial fermentation processes, product formation is always accompanied by biomass production. Since biomass formation, basically, goes at the expense of product formation, it would be beneficial to decouple growth from product formation in non-biomass driven applications. This project aims to investigate zero growth product formation with a genomics-based approach. In most industrial fermentations, the microorganisms will be present in this physiological state for the majority of the process

This project aims to investigate zero growth physiology in five different biological systems, baker’s yeast – Saccharomyces cerevisiae –, filamentous fungi (Aspergillus niger), Bacillus subtilis, the lactic acid bacterium, Lactobacillus plantarum, and the solvent-tolerant biocatalyst, Pseudomonas putida. This is done by studying the gradual changes that occur at the transcriptome and metabolome levels when the microorganisms go from a growing to a non-growing state. This transition is realized by retentostat cultivation, i.e. a cell-recycling fermentor where substrate is fed at a constant rate while cells are retained in the fermentor by means of an internal filtration device. In such a fermentation system, the biomass concentration, ultimately, reaches a steady-state where all substrate provided is used for maintenance purposes. Initial transcriptome analysis of S. cerevisiae, L. plantarum, and A. niger shows differential expression of several hundreds of genes under these “Zero Growth” conditions. In addition, clear differences in product formation, morphology, culture heterogeneity and robustness have been observed under these conditions. The study will provide a scientific basis for the rational improvement of product formation by this micro-organism at zero growth rate (i.e. without formation of excess biomass).



Karen McDonald

Ting-Kuo Huang1; Michael A. Plesha1; Bryce Falk2, Abhaya M. Dandekar3, and

Karen A. McDonald1

(1) Department of Chemical Engineering and Materials Science, University of California, Davis; (2) Department of Plant Pathology, University of California, Davis; (3) Department of Plant Sciences, University of California, Davis, One Shields Avenue, Davis, CA 95616

Plant cell suspension culture production of recombinant human therapeutic proteins provides unique advantages over traditional suspended prokaryotic (e.g. Escherichia coli) and eukaryotic (e.g. yeast, insect and mammalian cells) host expression cells in terms of product safety (plant cells and culture medium are not sourced from animals and do not harbor and propagate mammalian viruses), production cost (lower medium, production and downstream costs) and product quality (capability for protein post-translation modification). In this study, we investigated various strategies for improving the expression yield and functionality of a recombinant human blood protein, alpha-1-antitrypsin (AAT), using a novel plant viral amplicon expression system. The Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system was evaluated in transgenic tobacco (Nicotiana benthamiana) cell suspension cultures in a stirred-tank bioreactor.

For a chemically inducible plant viral expression system, the timing of induction (TOI) and concentration of inducer (COI) are two critical parameters for regulating the transgene expression in a chemically inducible cell culture process in bioreactor. We utilized the OUR (oxygen uptake rate) of plant cell cultures as a physiology indicator for determining the optimal TOI and developed a semicontinuous culture process for optimizing the COI effects. Total and functional recombinant AAT production yield, protease activity, total phenolics content, inducer (estradiol) concentration, OUR and biomass concentration were monitored as important parameters for bioreactor operation optimization.

One of the major findings of this work is the demonstration that long-term, steady state operation and continuous production of the recombinant extracellular recombinant protein production possible, even after the plant virus has been initiated and established in the cell culture. These results lay a foundation for developing efficient, scalable plant cell cultures as a platform for industrial biopharmaceuticals production.



Sathish Thadikamala

Production glutaminase by isolated bacterial strain, Bacillus sp., was studied using solid-state fermentation (SSF). Various locally available agro-industrial materials were collected, processed to segregate and used for fermentation experiments. Among all the materials studied, bengal gram husk showed effectiveness in production of glutaminase enzyme by isolated bacterial strain. In order to understand the impact of different substrate materials, SSF experiments were performed using mixed substrates (bengal gram husk, palm seed fiber and wheat bran). It was noticed that maximum glutaminase production was achieved using mixture of solid-substrate mixture at two-level combinations in the ratio of 66: 34 for bengal gram husk and wheat bran. Different fermentation parameters like particle size, pH of the medium, moisture content, inoculum level and glutamine concentration, were optimized during fermentation. Amylase was found to be a noise enzyme in this study which was found to decrease the production of glutaminase. In order to decrease noise enzyme production and increase glutaminase yield Taguchi experiments were performed. By employing the dynamic Taguchi design five fermentation controlling parameters, one noise parameter and one noise controlling parameter were optimized. The results indicated that optimization under noise factor controlled environment glutaminase production improved up to 247 %.







Moderator: Jeroen Hugenholtz, Kluyver Centre for Genomics of Industrial Fermentation (The Netherlands)

Presenter 1: A novel biosynthetic process concept for advanced ß-lactam antibiotics
A.J.M Driessen, University of Groningen, (Netherlands)  [Confirmed]

Presenter 2: Zero Growth Product Formation in Industrial Microorganisms 
Jeroen Hugenholtz, Kluyver Centre for Genomics of Industrial Fermentation, (The Netherlands)  [Confirmed]

Presenter 3: Synergistic Strategies to Improve the Microbial Enzyme Productivity in Solid State Fermentation - A Case Study 
Sathish Thadikamala, Indian Institute of chemical technology , (India)  []

Presenter 4 (if necessary): Development of Plant Cell Suspension Culture as a Bioproduction Platform For Recombinant Human Therapeutic Protein Production  
Karen McDonald, University of California, Davis, (United States)  [Confirmed]

Panel Organizer:
Matthew Carr, Biotechnology Industry Organization, (United States)

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