Characterization of pathway engineered strains of filamentous fungi in submerged cultures Promotor: Prof. dr. ir. J Tramper Hoogleraar in de Bioprocestechnologie Co-promotoren: M.Vetr.Sc., PhD JJL Iversen Associate professor, Department of Biochemistry and Molecular Biology, University of Southern Denmark Dr. GJG Ruijter Klinisch biochemisch geneticus io, Laboratorium Metabole Ziekten, Leids Universitair Medisch Centrum Dr. ir. J Visser Gastmedewerker Instituut voor Biologie, Universiteit Leiden Promotiecommissie: Cand. Ing. K Hansen (Novozymes A/S, Bagsvaerd, Denmark) Prof. dr. CAMJJ van den Hondel (Universiteit Leiden) Prof. dr. JT Pronk (Technische Universiteit Delft) Prof. dr. WM de Vos (Wageningen Universiteit) Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG. 2 Bjarne Rask Poulsen Characterization of pathway engineered strains of filamentous fungi in submerged cultures Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. ir. L. Speelman, in het openbaar te verdedigen op vrijdag 1 april 2005 des namiddags te half twee in de Aula 3 B.R. Poulsen - Characterization of pathway engineered strains of filamentous fungi in submerged cultures Ph.D. thesis Wageningen University, Wageningen, The Netherlands – with summary in Dutch ISBN 90-8504-154-6 4 Welcome Something totally beyond your control brought you here, into these pages: THE PRIMAL PULL OF WATER It resides in us all. Most fight against it’s tug only to live dry, predictable existences. Others, like you, concede the water's power and accept its embrace, asking only for a chance to explore its mysteries and feed a desire to understand how we fit into all of this. You've come to the right place. You are among friends. Wilderness Systems, North Carolina 5 6 Contents Chapter 1: General introduction 9 Chapter 2: Determination of first order rate constants by natural logarithm of the slope plot exemplified by analysis of Aspergillus niger in batch culture 13 Chapter 3: Homogeneous batch cultures of Aspergillus oryzae by elimination of wall growth in the Variomixing bioreactor 25 Chapter 4: Quantitative description of biomass distribution in free hyphae, pellets and diffusion-limited pellet cores in submerged cultures of filamentous fungi 41 Chapter 5: Fast response filter module with plug flow of filtrate for on-line sampling from submerged cultures of filamentous fungi 63 Chapter 6: Characterization of nerolidol biotransformation based on indirect on- line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger 81 Chapter 7: Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production 101 Chapter 8: Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger 115 Chapter 9: Can submerged cultures of filamentous fungi be made reproducible? A prerequisite for the omics technologies 145 Summary 165 Samenvatting 169 List of publications and patents 173 Curriculum Vitae 175 Thanks 177 7 8 Chapter 1 General Introduction Filamentous fungi are widely used in the industry for synthesis of antibiotics, biomass, enzymes and organic acids. Examples of products are penicillin, high-protein food (QuornTM), esterases as an additive in washing powder and citric acid in soft drinks. The domestication of filamentous fungi has a long history probably starting hundreds of years ago with production of sake with Aspergillus oryzae. Another important step was the start of production of citric acid with Aspergillus niger in the early 20th century, which at the beginning of the 21st century has reached a size of around a million tons per year [Ruijter et al. 2002]. The extensive use for food products has given A. niger its GRAS status (Generally Recognized As Safe, U.S. Food and Drug Administration), which is one of the reasons that this fungus was chosen for this study. Other reasons are the extensive knowledge that already exist about A. niger and that filamentous fungi efficiently produce and secrete homologous as well as heterologous proteins and execute post- translational modifications similar to higher eukaryotes. Strain improvement has so far been obtained mainly via classical methods, e.g. screening of mutants after radiation with UV. However, modern molecular techniques have made more direct approaches possible. The work described in this thesis presents such approaches and solutions to some of the problems encountered in growing and characterizing submerged cultures of filamentous fungi strains. Synthesis of most of the products mentioned above requires reducing equivalents in the form of NADPH. The pentose phosphate pathway (PPP) is a central pathway in primary metabolism (Fig. 1). The oxidative part of this pathway is believed to be the major source of NADPH required for many biosynthetic and detoxification reactions. In the non-oxidative part erythrose 4-phosphate and ribose 5-phosphate are generated for subsequent biosynthesis of aromatic amino acids and nucleotides, respectively. In addition, the polyols arabitol, erythritol and xylitol and several pentose sugars are metabolised via the PPP in fungi. There is an increasing interest to apply microorganisms in biotransformation processes for the synthesis of organic compounds. Microbial transformations have the advantage of proceeding under mild conditions preventing degradation of labile substrates or products. In addition, biotransformations are highly regio- and stereoselective conversions of complex organic molecules. A final advantage is that whole cell catalysts are inexpensive [Lehman and Stewart 2001]. Many processes are hydroxylations, oxidations or reductions done by fungal oxidoreductases probably in many cases involving cytochrome P450, which mostly require reducing power in the form of NADPH. For efficient biotransformation the supply of reducing equivalents should be sufficient and this might be accomplished by an elevated flux through the PPP. 9 Characterization of pathway engineered strains of filamentous fungi in submerged cultures Figure 1. Glycolysis, pentose phosphate pathway and polyol formation in Aspergilli. Partly after [Witteveen 1993] and [Ruijter et al. 2003]. Two arrows in series mean two or more reactions. Enzymes in boxes were subjected to metabolic engineering in this study. E and I indicate extra- and intracellular polyols, respectively. Metabolites and enzymes in italics were measured in wild type and engineered strains. See Chapter 8 for abbreviations. The aim of the work presented in this thesis is to increase the availability of NADPH and/or the flux through the PPP by metabolic engineering of A. niger wild-type and genetically modified strains. It might be possible to increase the flux through the PPP by overexpression of enzymes in the pathway or by disruption of genes in glycolysis. The objective is not merely to construct strains, but to do a thorough physiological investigation of wild type and transformant strains under various growth conditions by measurement of enzyme activities and concentrations of intracellular and extracellular metabolites at well-defined physiological states and during biotransformation. Such a comparison of wild type and modified strains requires reproducible culturing. This is particular challenging with filamentous fungi, since they have a strong tendency to adhere to surfaces and to form pellets and the interplay between these tendencies and physiology is very complicated. OUTLINE OF THE THESIS In Chapter 2 a method for analysis of exponential data is described. The natural logarithm of the slope (LOS) plot enables identification of (1) data intervals that develop exponentially, (2) the rate constant (e.g. the growth rate) and (3) small and/or sudden changes in the rate constant. A bioreactor specially designed for growth of filamentous fungi is described in Chapter 10 Chapter 1 General introduction 3. In this “Variomixing” bioreactor, wall growth is kept to a minimum by computer-controlled rotation of the baffles at a similar speed and direction as the impeller for 5 sec every 5 min, which results in a temporary cancellation of the effect of the baffles giving a deep vortex and high peripheral liquid flow rates at the reactor wall. The “Macro-morphology Profiling System” (MPS) described in Chapter 4 is a quantitative description of the amount of biomass in free hyphae and in pellets with core diameters smaller or larger than the critical core diameter, defined as the largest pellet core diameter without diffusion limitation of substrate in centre. In addition, MPS describes the amount of biomass limited by substrate diffusion into pellet cores larger than the critical core diameter. It was used to study the relationship between macro-morphology and physiology in batch, washout and chemostat cultures. Invention and test of a filtrate sampling unit with a fast response time is given in Chapter 5. A response time of around 1 min for sampling of 1 mL⋅min-1 was obtained, enabling automatic sampling sufficiently accurate to monitor even glucose pulse experiments. Biotransformation of nerolidol to hydroxy-nerolidol by A. niger is described in Chapter 6. The influence of physiological state and growth conditions on biotransformation and yield was determined. In Chapter 7 optimization of morphological stability in the chemostat cultures (Chapter 4) is described. An apparently stable aconidial (fluffy)