Primary Metabolite Production

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Shanna Gibbs
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1 Primary Metabolite Production During the exponential phase organisms produce a variety of substances that are essential for their growth, such as nucleotides, nucleic acids, amino acids, proteins, carbohydrates, lipids, etc., or by- products of energy yielding metabolism such as ethanol, acetone, butanol, etc. These products are usually called primary metabolites. Commercial examples of such products : Primary Metabolite Organism Significance tons per year Ethanol Saccharomyces cerevisiae alcoholic beverages Kluyveromyces fragilis 26,000,000 Citric acid Aspergillus niger food industry 1,000,000 Acetone and butanol Clostridium acetobutyricum solvents Lysine Corynebacterium nutritional additive 350,000 Glutamic acid glutamacium flavour enhancer 1,000,000 Riboflavin Ashbya gossipii Eremothecium nutritional ashbyi Vitamin B12 Pseudomonas denitrificans nutritional 10 Propionibacterium shermanii Dextran Leuconostoc mesenteroides industrial Xanthan gum Xanthomonas campestris industrial ,000

2 Biomass and Ethanol + biomass

3 Biomass and Ethanol ethanol

4 Biomass and Ethanol ethanol + biomass

5 Net balance of fermentation to ethanol: C 6 H 12 O 6 2 C 2 H 5 OH + 2 CO 2 glucose 2 ethanol + 2 carbon dioxide Energy balance: C 6 H 12 O P i + 2 ADP 2 C 2 H 5 OH + 2 CO ATP

7 Redox Balance One essential feature of cellular metabolism is a closed redox balance. Glycolysis is an oxidative process, leading to the accumulation of reduced NADH. The cell has to reoxidize NADH to keep glycolysis active. This is achieved either by the respiration chain, or by the formation of reduced metabolites (ethanol, lactate, glycerol etc.).

9 (see figure before)

10 Pasteur-Effect: Aerobic conditions lead to a reduction of the sugar consumption and the fermentation. Was observed with stationary Saccharomyces cerevisiae, and is NOT VALID for active S. cerevisiae cultures!! "Crabtree-Effect": In the strict sense: Inhibition of respiration through glucose (observed in tumours). This term is used for yeasts, when most of the glucose is metabolized fermentatively even under aerobic conditions. Better: "Aerobic Fermentation", or "Aerobic Ethanol Formation"

12 Energy yields Respiratory glucose consumption: 1 Glucose 38 ATP Respiratory ethanol consumption: 1 Ethanol 11 ATP Glucose fermentation to ethanol: 1 Glucose 2 ATP + 2 Ethanol Sum glucose fermentative-aerobic: 1 Glucose 24 ATP

13 Metabolic types of yeasts Facultatively anaerobic yeasts (Facultatively fermentative) Obligatory aerobic yeasts (non fermentative) Fermentative yeasts ( Crabtree positive ) Respiratory yeasts ( Crabtree negative ) e.g. Rhodotorula, Cryptococcus e.g. Saccharomyces, Schizosaccharomyces, Brettanomyces e.g. Candida Hansenula Pichia No fermentative metabolism possible Glucose consumption high ( µmol g -1 min -1 ) O 2 -consumption low (5-50 µmol g -1 min -1 ) > 90 % fermentative Glucose consumption low (10-40 µmol g -1 min -1 ) O 2 -consumption high ( µmol g -1 min -1 ) max. 30 % fermentative

14 Conclusion concerning energy and biomass yield Fermentative yeasts consume glucose appr. 10x faster During fermentative metabolism the energy yield per unit glucose is appr. 1/20 compared to aerobic metabolism BUT: due to the much faster uptake, there is almost no difference in total energy production rate AND: the energy stored in glucose is not wasted for the yeast: it may be metabolized later aerobically from ethanol

15 Glucose consumption is controlled by glucose uptake Kinetics of glucose uptake systems (summary of literature values) species uptake type Km [mm] v max [µmol/g min] S. cerevisiae low affinity high affinity S. carlsbergensis low affinity high affinity 3 20 S. bayanus low affinity high affinity P. guillermondii low affinity high affinity Y. lipolytica low affinity high affinity E. coli pts IIGlc pts IIMan GalP Attention: Values depend on culture type!

16 Glucose transporters of yeasts S. cerevisiae encodes 20 hexose transporters. They work via facilitated diffusion. Some have low affinity to glucose, others high affinity. They are regulated by glucose: High glucose concentration induces low affinity transporters (e.g. Hxt1and Hxt3). Low glucose concentration induces high affinity transporters (e.g. Hxt2, Hxt6, Hxt7). All other yeasts have less glucose transporters, but usually both high and low affinity transporters.

17 The phosphoenolpyruvate:sugar phosphotransferase system and other glucose transport systems in Escherichia coli.

18 How to derive specific growth rates from substrate uptake kinetics Substrate uptake is controlled by the kinetics of the resp. uptake system: In an idealized organism we can assume one uptake system characterized by its kinetic parameters K m and v max. They are related by a Monod type equation: v i = v max ( c S i c S + i K m ) Growth depends on substrate uptake and biomass yield µ = v Y ' i i XS

19 Glucose uptake rates and specific growth rates (values derived from chemostat) Species v max/glc [g/g h] µ max [1/h] Y X/S (calc) S. cerevisiae resp. 0,36 0,2 0,55 S. cerevisiae ferm. 3,0 0,45 0,15 E. coli 2,8 1,4 0,5 P. pastoris 0,36 0,2 0,55 v glc [g/g h] = v glc [µmol/g min] / 10 6

20 Reality is more complex! In reality one has to consider the different glucose transporters with different kinetics and different regulation. In steady state (e.g. chemostat or fed batch with exponential feed) one transport system will dominate (the other being at low activity due to kinetics and regulation). BUT: in transient situations such as batch cultures, changing glucose uptake has to be considered. Substrate uptake kinetics is also essential for mixed cultures!

21 Conclusions from glucose uptake kinetics Among the yeasts listed, S. cerevisiae and S. carlsbergensis consume glucose at high velocity in glucose rich environment (Km=100 mm means, at 18 g/l the glucose uptake rate is half maximal). At glucose concentrations below 0,2-0,5 g/l the glucose uptake rate (and growth rate) are significantly retarded. Other yeasts have lower maximal glucose consumption rates (and growth rates), but maintain the glucose consumption rate at low glucose concentrations (0,02 g/l). E. coli combines very high glucose uptake rates with the ability to maintain them at very low glucose concentrations (0,001-0,002 g/l).

22 Maximum glucose consumption rates determine: Maximum specific growth rates (µ) Maximum specific product formation rates (q P ) Maximum volumetric productivities (Q P )

23 Back to aerobic fermentation: S. cerevisiae in chemostat at different dilution rates (= µ) Dry mass, glucose and by-products at different µ

24 Regulation through substrate affinity pyruvate pyruvate decarboxylase PDC acetaldehyde alcohol dehydrogenase ADH ethanol pyruvate dehydrogenase complex PDH acetylcoa acetaldehyde dehydrogenase ALD acetate TCA cycle K m(pdh) : K m(pdc) 1:10 for pyruvate K m(ald) : K m(adh) 1:100 for acetaldehyde

25 Low [pyruvate] TCA cycle High [pyruvate] acetaldehyde Low [acetaldehyde] acetate High [acetaldehyde] ethanol The concentrations of pyruvate and of acetaldehyde correspond with the resp. fluxes High glycolytic flux most pyruvate goes through PDC conc. of acetaldehyde increases most flux through ADH At high glycolytic flux ADH reaction is needed to recycle oxidized NAD + When glucose uptake is strictly controlled (low vmax) low pyruvate conc. (almost) no fermentation

26 Also enzyme activity depends on glucose flux ADH1 induced by glucose repressed by ethanol (acetaldehyde ethanol) ADH2 repressed by glucose induced by ethanol (ethanol acetaldehyde) similar: several PDC isoenzymes

27 Absolute carbon fluxes of S. cerevisiae in chemostat under aerobic glucose-limited conditions at different growth rates: A: cytosolic B: mitochondrial For each enzyme the flux at purely oxidative (µ = 0.15 h -1 ), respiro-fermentative (µ = 0.30 h -1 ), and mainly fermentative growth (µ = 0.40 h -1 ) is shown from left to right. From: Frick and Wittmann, Microbial Cell Factories 2005, 4:30

28 Intracellular carbon flux distribution of S. cerevisiae cultivated in chemostat on [1-13 C] glucose under aerobic glucose-limited conditions at different growth rates. All fluxes are given as relative fluxes normalized to the specific glucose uptake rate. For each reaction the fluxes corresponding to purely oxidative (µ = 0.15 h -1, q glc = 1.56 mmol g -1 h -1 ), respirofermentative (µ = 0.30 h -1, q glc = 4.90 mmol g -1 h -1 ), and mainly fermentative growth (µ = 0.40 h -1, q glc = 8.23 mmol g -1 h -1 ), respectively, are shown from top to bottom. For reversible reactions an additional arrow indicates the direction of the net flux and the values in the squared brackets are the obtained reversibilities of the corresponding enzymes. The fluxes correspond to the optimal fit between experimentally determined steadystate 13 C labeling patterns of amino acids of the cell protein and 13 C labeling patterns simulated via isotopomer modelling. From: Frick and Wittmann, Microbial Cell Factories 2005, 4:30

29 Conclusions from Flux analysis Glycolytic flux to pyruvate correlates strongly with glucose uptake rate. Mitochondrial fluxes (TCA cycle = Zitratzyklus) are rather constant throughout. The higher glycolytic flux at high glucose uptake rates is channelled to fermentation: mainly acetate and ethanol.

30 Mixed culture of Crabtree positive and negative yeast at glucose limit Saccharomyces cerevisiae Candida utilis

31 Conclusions Crabtree positive yeasts (microorganisms) have an advantage in substrate rich environments (high sugar concentration): They consume the sugar at high rate (much higher than TCA cycle is able to turn over). They convert it to a toxic by-product (e.g. ethanol, lactate; more toxic to competitors). They can metabolize the by-product later with an overall energy yield similar to direct oxidative metabolism. Crabtree negative yeasts have an advantage in substrate limited conditions (poor environments): The sugar uptake is dominated by high affinity transporters (usually at lower rate). Therefore they consume most of the sugar in low concentration environments.

32 Schematic flow-chart of ethanol production

35 biomass and ethanol + biomass

36 Baker's yeast production In 1847, the Federation of Industry of Lower Austria offered a reward for a process to produce baker's yeast from grain. The competition was won by Julius Reininghaus, who started a business with A. Mautner. This process achieved world reputation as the "Vienna Process".

37 Baker's yeast production Around 1920, the Vienna Process was replaced by a modern process involving aeration and controlled feeding of the nutrients, called the "Zulaufverfahren". Alcoholic fermentation is limited by a limitation of carbon source.

38 Ausbeuten aus 100 kg Getreide Vergleich der alten Verfahren: Verfahren kg Hefe Liter Alkohol Holländisches Verfahren Wiener Verfahren Hefe-Lüftungsverfahren Zulaufverfahren 85 1

39 The "Zulaufverfahren" today The "Zulaufverfahren" is not only used for baker's yeast production, but, as the fed-batch process, it is the basis for most modern biotechnology processes for the production of biopharmaceuticals.

40 Fed batch means controlled addition of substrate to the fermenter without removal of culture broth Aim: reduction of available substrate to avoid overflow metabolism Simple scheme of fed batch Fed batch fermentation with process control

41 (a) V (c) V F µ µ F time time (b) V F µ time! The volume is not constant in fed batch!

43 Schema der Backhefe- Fermentation

45 Beispiel: Schema einer Backhefe-Produktionsanlage

46 Single Cell Protein In the 1970ies, several large projects were developed to produce yeast protein as feed (or human food). One plan was to use hydrocarbons as carbon source. This led to the development of large scale fermentation processes of methylotrophic yeasts, like e.g. Pichia pastoris. These yeasts can metabolize C1 substrates like methanol (which is obtained from methane by oxidation). Additionally, these yeasts are Crabtree negative, which means that biomass can be produced by simple batch processes. Today, methylotrophic yeasts play an important role as hosts for heterologous protein production.

Chemical Energy. Valencia College

Chemical Energy. Valencia College 9 Pathways that Harvest Chemical Energy Valencia College 9 Pathways that Harvest Chemical Energy Chapter objectives: How Does Glucose Oxidation Release Chemical Energy? What Are the Aerobic Pathways of