Industrial Biotech LearningGoals PDF

00a_General Introduction to Industrial Biotechnology 1) Reflect the status quo of the current chemical industry in terms of sustainability (raw materials/carbon sources, catalysts, energy consumption, waste streams) 2) Remember from 1st year lecture: What is industrial (white) biotechnology? White...

00a_General Introduction to Industrial Biotechnology 1) Reflect the status quo of the current chemical industry in terms of sustainability (raw materials/carbon sources, catalysts, energy consumption, waste streams) 2) Remember from 1st year lecture: What is industrial (white) biotechnology? White biotechnology is arguably the largest area of biotechnology. It revolves primarily around the use of biocatalysts for the industrial-scale production and processing of products. There is also a focus on reducing the environmental impact of industrial processes, involving the production of biodegradable polymers and renewable fuel to encourage a more sustainable system. The use of renewable resources instead of fossil resources is another core aspect of industrial biotechnology. Why does the chemical industry need to change and why is it promising to include Biotechnology? Traditional chemical production processes are often not suitable for the utilization of renewable sources of carbon. Biotechnology is versatile and has been assessed as a key technology for a sustainable chemical industry. Industries that previously never considered biological sciences as impacting their business are exploring ways of using biotechnology to their benefit. Biotechnology provides entirely novel opportunities for sustainable production of existing and new products and services. Environmental concerns help drive the use of biotechnology in industry, to not only remove pollutants from the environment, but prevent pollution in the first place. Biocatalyst-based processes have major roles to play in this context. Biocatalysis operates at lower temperatures, produces less toxic waste, fewer emissions and by-products compared to conventional chemical processes. New biocatalysts with improved selectivity and enhanced performance for use in diverse manufacturing and waste degrading processes are becoming available. In view of their selectivity, these biocatalysts reduce the need for purifying the product from byproducts, thus reducing energy demand and environmental impact. Unlike non-biological catalysts, biocatalysts can be self-replicating. 3) What is meant by fossil carbon? Carbon that is stored in fossil fuels. Fossil fuels are hydrocarbon-based energy sources formed from the remains of ancient plants and organisms that lived millions of years ago. These fuels include coal, oil (petroleum) and natural gas. Fossil carbon represents the carbon that has been sequestered in these fuels over geological time periods. 4) What are renewable sources of carbon? Renewable carbon sources are carbon-containing materials or compounds derived from sustainable and regenerative processes, as opposed to fossil carbon sources, which are finite and non-renewable. These renewable sources of carbon play a vital role in addressing environmental and sustainability challenges, including reducing greenhouse gas emissions, promoting circular economy principles, and mitigating climate change. Here are some examples of renewable carbon sources: Biomass is one of the most prominent renewable carbon sources. It includes organic materials derived from plants, trees, and agricultural crops, as well as organic waste materials. Biomass can be used to produce renewable energy, such as biofuels (e.g., biodiesel and bioethanol), or to generate heat and electricity. Biochar is a type of charcoal produced through the pyrolysis of biomass (organic matter) under controlled conditions. It can be used to improve soil quality and sequester carbon in the soil, promoting carbon capture and storage (CCS) in a sustainable manner. Crop residues, such as straw, stalks, and husks, can be used as renewable carbon sources for various applications, including bioenergy production, animal feed, and soil improvement. Algae are a renewable source of carbon that can be grown in aquatic environments. They can be used for the production of biofuels, such as biodiesel, and for capturing CO2 from industrial emissions. 5) Enzymes are used as catalysts in order to conduct chemical reactions and produce chemicals of interest. In industrial biotechnology, the enzymes/catalysts can either be used in an isolated form (= cell-free) or stay inside of cells. 6) Industrial biotechnology mainly makes use of microorganisms and their ingredients/enzymes (rather than plant or animal cell lines which are barely used). 7) Explain which characteristics of the yeast Saccharomyces cerevisiae have led to the fact that this microorganism became an important cell factory in industrial biotechnology? - ​ Eukaryote (model organism with traditional use in large-scale fermentations). - ​ Robust (pH, alcohols, inhibitors). - ​ Crabtree positive=high glucose concentrations suppress aerobic respiration in favor of fermentation, even in the presence of oxygen - ​ Facultative anaerobe. - ​ Accessible to extensive metabolic engineering. 01 INTRODUCTION TO BIOCATALYSTS 1) What is catalysis (in general) and what is a catalyst in chemistry? Catalysis is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst, which is not consumed in the catalyzed reaction and can continue to act repeatedly. Because of this, only very small amounts of catalyst are required to alter the reaction rate. Chemical reactions occur faster in the presence of a catalyst because the catalyst lowers the activation energy required for the reaction to begin. In catalyzed mechanisms, the catalyst usually reacts to form a temporary intermediate, which then regenerates the original catalyst in a cyclic process. ​Catalysis is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst, which is not consumed in the catalyzed reaction and can continue to act repeatedly. 2) What is an enzyme? Make the connection between „enzyme“ and „biocatalyst“. Enzymes are proteins and proteins are encoded by protein-encoding genes. Enzymes are the catalysts in nature (inside the cells and outside the cells); they make up the metabolic pathways/metabolism of cells. 3) Why does a cell need „extracellular enzymes“? Do you know examples? Cells need extracellular enzymes to break down large molecules in their environment into smaller, absorbable components. These enzymes are secreted outside the cell and act in the surrounding medium to facilitate the digestion or processing of complex substrates. Examples of Extracellular Enzymes: 1.​ Amylase: Breaks down starch into maltose and glucose. 2.​ Protease: Degrades proteins into peptides and amino acids. 3.​ Lipase: Hydrolyzes fats into glycerol and fatty acids. 4.​ Cellulase: Decomposes cellulose into glucose units (important for decomposing plant material). 5.​ Laccase: Involved in lignin degradation. These enzymes are crucial for organisms like fungi and bacteria that rely on external digestion to acquire nutrients. In industrial biotechnology, extracellular enzymes are often utilized for processes like fermentation, biotransformation, and biocatalysis​ 4) Definitions of the terms (mainly recapitulation): metabolite, metabolic pathway, metabolism, biomass, metabolomics, enantiomers, chiral center. Here are the definitions for the requested terms, based on biochemistry and industrial biotechnology: 1. Metabolite ​ A metabolite is a product, intermediate, or substrate of enzymatic reactions in metabolism. These are typically small molecules involved in the processes of energy generation or biosynthesis. ​ Examples: Glucose, pyruvate, ATP. 2. Metabolic Pathway ​ A metabolic pathway is a sequence of enzymatic reactions where the product of one reaction serves as the substrate for the next. These pathways can be: ○​ Catabolic: Breaking down complex molecules (e.g., glycolysis). ○​ Anabolic: Synthesizing complex molecules (e.g., fatty acid synthesis). ○​ Amphibolic: Functions in both breakdown and synthesis (e.g., the citric acid cycle). 3. Metabolism ​ Metabolism encompasses all the chemical reactions in a cell or organism, including the synthesis (anabolism) and breakdown (catabolism) of molecules to maintain life. It involves energy transfer and the generation of metabolites. 4. Biomass ​ Biomass refers to the total mass of living or organic material within a specific environment or system. In biotechnology, it often refers to the microbial or cellular mass generated during fermentation or growth processes. 5. Metabolomics ​ Metabolomics is the study of the complete set of metabolites (metabolome) present in a biological system under specific conditions. It provides insights into the metabolic state of an organism or cell. 6. Enantiomers ​ Enantiomers are a pair of molecules that are non-superimposable mirror images of each other, often differing in their biological activity or properties. ​ Example: (S)-limonene smells like oranges, while (R)-limonene smells like lemons. 7. Chiral Center  ​ A chiral center is an atom, typically carbon, that is bonded to four different groups, creating two non-superimposable mirror image forms (enantiomers). Chirality is essential in biochemistry for specificity in enzyme interactions. These terms are foundational in understanding enzymatic processes and their applications in biotechnology. 5) What is biocatalysis? What do we call a biocatalyst? Biocatalysis Biocatalysis refers to the use of natural catalysts, such as enzymes or whole cells, to perform chemical reactions. It is a process where biological systems accelerate the transformation of substrates into desired products, often with high specificity, efficiency, and under mild conditions. ​ Examples of Biocatalysis Applications: ○​ Synthesis of pharmaceuticals. ○​ Production of biofuels. ○​ Environmental cleanup (bioremediation). Biocatalyst A biocatalyst is any biological molecule or system that facilitates a chemical reaction. These can be: 1.​ Cell-Free Biocatalysts:​ ○​ Isolated enzymes: Purified enzymes used to catalyze specific reactions. ○​ Extracellular enzymes: Enzymes naturally secreted by cells into the environment. 2.​ Whole Cells:​ ○​ Microorganisms: Cells containing multiple enzymes that can perform multi-step transformations. Biocatalysts are valued for their: ​ High reaction rates (accelerating reactions by factors of 10⁸–10¹²). ​ Selectivity (chemo-, regio-, and stereoselectivity). ​ Functionality under mild and environmentally friendly conditions. 6) Which characteristics make biocatalysis attractive (compared to chemical catalysis)? - ​ Enzymatic reations show a high efficiency (enzymes accelerate the rates of chemical reactions by factors of 108 to 1012).  - ​ Enzymatic reactions show a high specificity (e.g. stereoselectivity allows for the production of the enantiomer in excess=enantiomeric excess. In chemical catalysis, you can only produce the two compounds without specificity). - ​ Enzymes/cells work at ambient temperature, away from pH extremes, in aqueous solutions and under atmospheric pressure (i.e. more environmentally friendly and less energy-intensive). - ​ Often, multi-step chemical conversions can be replaced by a single enzymatic reaction. - ​ Undesired isomerization, racemization, epimerization and rearrangement reactions frequently encountered during chemical processes are generally avoided (i.e. less side product formation = lower costs for product purification). - ​ Many natural valuable natural compounds/chemicals cannot be degraded (e.g. cellulose) or produced (e.g. artemisinin) by chemical catalysis (i.e. without biocatalysts). 7) Be aware of the difference between cell-free biocatalysts (isolated/cell-free or extracellular enzymes) and entire cells (containing multiple enzymes that manage cellular metabolism) – both can be considered as „biocatalysts“ depending on the author. 8) What are the global driving forces for the increasing implementation of industrial biotechnology in chemical industry? The global driving forces for the increasing implementation of industrial biotechnology - ​ Growing rate of energy consumption. → IBT: Lowered energy consumption (due to lower reaction temperatures). - ​ Future depletion of non-renewable (fossil) resources. → IBT: Use of renewable resources such as plant biomass (produced from CO2 and light by photosynthesis). - ​ Global climate change. → IBT: Lower greenhouse gas emissions due to better carbon balance. - ​ Omnipresence of human-generated toxic waste. → IBT: Biodegradable products and less toxic waste. - ​ Strongly increasing market prices for fossil resources. → IBT: Cheaper production combined with improved product quality/purity, due to significant performance benefits compared with conventional chemical technology such as higher reaction rate, increased conversion efficiency and specificity (unfortunately, this does not always apply). What is the difference between fermentation (de novo biosynthesis) and biotransformation? In the exam! Fermentation (de novo biosynthesis): Relies on growing cells which have an active metabolic network. Multistep conversions using the cell’s metabolism and growing cells. It is a metabolic process primarily aimed at producing energy and metabolites (end products) for the microorganism’s own growth and survival. The cells need biomass (cell wall, cell membrane etc constituents), so they perform de novo biosynthesis utilizing nutrients (substrates, which come from breaking down complex organic compounds) to synthesize components they need to grow (products of metabolism). During fermentation microorganisms break down complex organic compounds into simpler substances, generating ATP for their energy needs. Example process: bioethanol production. Starch is broken down into sugars, glycolysis then alcoholic fermentation. Biotransformation: A metabolic process that transforms/converts one chemical compound (educt) into another chemical compound (product). Single (can also be multiple) step conversions using cell-free enzyme(s) or whole (most often non-growing) cells. Often used for specific applications, such as the production of valuable chemicals, pharmaceuticals or the modification of existing compounds. The primary purpose of biotransformation is not necessarily to generate energy but rather to create new compounds or modify existing ones. 02 HISTORICAL MILESTONES DURING THE DISCOVERY OF ENZYMES (INSIDE AND OUTSIDE OF CELLS) 1) What was meant (in history) by the terms „organized ferment“ and „non-organized ferment“? Do you know examples for both? At the time of their discovery, enzymes were named ferments. They were either “non-organized” or “organized”. “Organized ferments“ are cells (microorganisms) and “non-organized ferments“ are enzymes (disruption of cells or naturally secreted by the cell). Organized ferment examples: sugar fungus (Saccharomyces cerevisiae). Non-organized ferment examples: Pepsin (digestive enzyme found in the stomach that can be isolated and used in the food industry), cellulase, lipase, and diastase. 2) What did the historical terms „diastase“ and „zymase“ mean? In the early nineteenth century, the degradation of any kind of compound due to the action of another was generally described as fermentation. In 1833, Anselme Payen and Jean François Persoz isolated what they called a starch-liquefying principle (soluble/non-organized ferment) from crude extracts of germinated barley. Comparatively small amounts of the preparation were able to turn large quantities of starch into liquid. This ability was lost when exposed to heat. This “biocatalyst“ was clearly different from chemical catalysts. The described compound was named “diastase“ and was the first plant ferment (nowadays called “enzyme”) to be purified and studied in a laboratory. Today, diastase means a mixture of different amylases that can break down starch into sugars. Non-organized ferments: Diastase, pepsin that is naturally secreted by cells. Today we call non-organized ferments cell-free enzymes. Zymase: Initially thought to be the yeast enzyme responsible for the conversion of glucose to ethanol and carbon dioxide. In reality they are yeast enzymes catalyzing 12 chemical conversions. 3) How did Louis Pasteur disprove the hypothesis of „sponaneous generation“? Louis Pasteur disproved the hypothesis of spontaneous generation through his famous swan-neck flask experiment in 1860. The spontaneous generation theory suggested that living organisms could arise from non-living matter spontaneously. Pasteur's Experiment: 1.​ Preparation:​ Pasteur took two swan-neck flasks, both containing a nutrient-rich broth, and boiled them to sterilize the contents, killing any existing microorganisms.​ 2.​ Experimental Design:​ ○​ One flask was left with its long, curved neck intact, preventing dust and microbes from reaching the broth but allowing air to enter. ○​ The other flask had its neck broken, exposing the broth to airborne particles.  3.​ Observations:​ ○​ The broth in the intact swan-neck flask remained clear and free of microbial growth. ○​ The broth in the flask with the broken neck became cloudy, indicating microbial contamination. 4.​ Conclusion:​ ○​ Microbial growth occurred only when airborne microorganisms were allowed to enter the broth, proving that life did not spontaneously generate in sterilized conditions. Microorganisms came from the environment. This experiment not only refuted spontaneous generation but also established the principle of biogenesis: life arises from pre-existing life. 4) How did Eduard Buchner prove that the enzymes inside yeast are responsible for the alcoholic fermentation? He broke down yeast cells and saw that the cell-free enzymes in the extract were able to catalyze the reactions. In the early nineteenth century, it was generally assumed that fermentation is a chemical conversion from the substrate into ethanol and carbon dioxide brought about by a particular compound (present in grapes) which was called a ferment. 03 BIOPROCESSES PART A 1) Bioprocesses make use of biocatalysts (enzymes and cells) in many different ways; there are processes using cell-free enzymes while others use whole cells. 2) There are two different concepts of using whole cells: -​ in one concept (whole cell biotransformation), we use one (or a few) enzymes inside the cells as a catalyst -​ in a second concept (de novo biosynthesis), we grow cells by providing all required nutrients and, at the same time, use the metabolic network to produce a valuable chemical 3) Clarify terms: biotransformation, de novo biosynthesis, fermentation, whole-cell biotransformation. 1. Biotransformation ​ Biotransformation refers to the chemical modification of a compound by a biological agent, such as enzymes or microorganisms. This is typically used to convert substrates into more valuable or functional products. ​ Example: Conversion of proline into hydroxy-L-proline using enzymes. 2. De Novo Biosynthesis ​ De novo biosynthesis is the process where cells construct complex organic molecules from simple starting materials (such as sugars or amino acids) through metabolic pathways. ​ Example: Synthesis of vinblastine precursors from sugars by engineered Saccharomyces cerevisiae. 3. Fermentation ​ Fermentation is a metabolic process in which microorganisms (or enzymes) convert organic compounds, such as sugars, into simpler compounds, often producing energy in the absence of oxygen. ​ Types: ○​ Alcoholic Fermentation: Conversion of glucose to ethanol and CO₂ by yeast. ○​ Lactic Acid Fermentation: Conversion of glucose to lactic acid by bacteria. 4. Whole-Cell Biotransformation ​ Whole-cell biotransformation involves using intact, living cells as biocatalysts. The cells perform transformations using their metabolic pathways and enzymes. ​ Advantages: Whole cells can carry out complex, multi-step reactions that isolated enzymes cannot. ​ Example: Using E. coli to produce complex pharmaceutical precursors. 4) Become aware that the use of the terms ‚biotransformation‘, ‚biocatalysis‘ and ‚fermentation‘ is not consistent in literature (How do we define these terms throughout the lecture?) Different types of biotransformations exist: 1) ​ Use an enzyme naturally secreted by cells. 2) ​ Disrupt cells and purify the enzyme(s) of interest. 3) ​ Use crude cell extract. 4) ​ Use whole cells containing the enzyme of interest (in addition to thousands of other enzymes) = whole cell biotransformation. 5) Know what the term fermentation means in a strict and in a broad sense Fermentation in the broad sense: In biotechnology, fermentation is a biological production process (bioprocess) which uses growing cells (most often microorganisms) and starts from simple nutrients and generates intermediates or products of the cell‘s metabolism. Fermentation in a strict (biochemical) sense: Fermentation is a biochemical process that takes place in order to regenerate NAD+ for glycolysis in the absence of oxygen (which is the final electron acceptor of the electron transport chain). In the absence of an external electron acceptor, the organism has to sacrifice energy-rich compounds (such as pyruvate) as electron acceptor in order to gain the 2 mol ATP obtained from glycolysis → cells are forced to produce a waste (fermentation) product such as lactic acid. Bioethanol production. 6) Immobilization of biocatalysts (enzymes or cells) has certain advantages The point of immobilized enzymes is that they are recyclable. They are bound to large carriers which are visible to the naked eye and can be separated mechanically from the reaction solution. Enzymes can be bound to their carriers by chemical (covalent bond) or physical (adsorption or electrostatics forces) means. Special reagents are used to cross-link enzyme molecules, or they are mechanically entrapped by gel or hollow fibers. The production of immobilized enzymes for industrial purposes must be simple and cheap. Enzyme activity must be high in relation to the carrier mass, and the enzymes must be very stable within their action range. Immobilized enzymes are used in various types of enzyme reactors, most of which are either column reactors or stirrer vessels. Advantages: - ​ The immobilized enzymes are recyclable (reduced costs). - ​ They have the desired chemical and physical properties and often also higher stability in a wider pH range and at higher temperatures. - ​ The end product is free of enzyme residues. - ​ Easy product purification (downstream processing) due to easy separation of the product from the biocatalyst. - ​ Facilitates a continuous mode of operation - ​ Might provide a protective microenvironment to the biocatalyst resulting in a more stable catalyst. 03 BIOPROCESSES PART B 1) The advantages of using bioreactors instead of microtiter plates or shake flasks for conducting bioprocesses Large-scale bioprocesses are conducted in bioreactors. They are equipped with certain components to control the bioprocesses. Bioreactors can grow microorganisms, conduct bioprocesses. -​ The oxygen cannot optimally go in the solution then in the microorganisms in microtiter plates and Erlenmeyer flasks. You cannot shake so much. Oxygen transfer from gas phase to the liquid phase is not optimal. You can continuously bring oxygen in bioreactors. In bioreactors there is a mixer in there that optimally delivers oxygen to the microorganisms. -​ Mixing is not optimal. -​ In a bioreactor you can continuously feed medium, while in a microtiter plate and Erlenmeyer flask you have to go to the clean bench etc. Very time consuming and not 100% sterile. Constant medium feeding is not possible. -​ pH cannot be controlled. Can control the pH in bioreactors. pH is important because there is an optimum pH range where the enzyme works best. pH is changing due to the growing cells’ metabolism (e.g., ATPases that pump protons out of the cell to facilitate uptake of nutrients). -​ The consumption/production of gases (e.g. O2, CO2) cannot be recorded. The throughput (how many samples can be analyzed at a time) is much higher for microtiter plates. Steam vessel allows sterilization after the process is done. Exhaust allows the release of gases (CO2 produced during alcoholic fermentation along with ethanol) that would normally increase the pressure inside. Cooling jacket: for example, during glycolysis a lot of heat is produced. Cells could die. 2) The different types of feeding regimes: batch, fed-batch and continuous cultivation The different process modes of operation are often referred to as “fermentation modes“ such as batch fermentation, fed-batch fermentation, continuous fermentation. Batch operation: -​ Bioreactor is filled with medium up to maximal volume, no additional medium feeding. -​ Microorganism is inoculated. -​ Culture behaves according to a typical growth curve in batch. Advantages: -​ Easy and robust. -​ Low risk of contamination. Disadvantages: -​ Substrate/product inhibition possible. -​ Osmotic stress at the beginning of the process (high glucose conc – water goes out of the microorganism and it shrinks). -​ Frequent cleaning/sterilization required (i.e. after each harvest). Fed-batch operation: -​ Start as batch process with 1/3 to ½ of maximal volume. -​ When substrate is almost consumed, feeding with fresh medium starts. -​ Aim is to keep low (and preferably constant) substrate concentration. Advantages: -​ Reduces substrate/product inhibition. -​ Keeps the cells exponentially growing for a longer time period. -​ Prevents osmotic stress. -​ Higher product titers (= concentrations) possible (if substrate) Disadvantages: -​ More laborious than a batch operation. Continuous operation (chemostat): In a continuous mode of fermentation you can control the rate of growth by addition/removal of limiting nutrient. -​ Start as batch or fed-batch process. -​ When substrate is almost consumed/max. volume is reached, continuous feeding (inlet) AND outlet flow is simultaneously set. -​ Aim is to keep LOW and CONSTANT substrate concentration = Growth rate (μ) can be adjusted by flow rate = cells are in steady state. Advantages: -​ Prevents substrate/product inhibition. -​ Keeps the cells growing in the exponential phase (= steady state). -​ Prevents osmotic stress. Disadvantages: -​ Laborious. -​ Higher risk of contamination. -​ Lower product concentrations (in the outlet). 3) Understand (and be able to calculate) the measures titer, yield and production rate 4) Make a difference between volumetric and specific production rate Volumetric rate does not take into account how many biocatalysts you have in the solution. It does not tell you anything about the performance of a single cell or a strain or a microbe. Overall process. Dilution rate: The rate at which you add the growth-limiting nutrient. This way, you can control the growth rate of the microorganisms. 5) Maximum theoretical yield=refers to the highest possible amount of a desired product that can be obtained from a given amount of reactants, based on stoichiometric calculations, assuming 100% efficiency and no losses. It represents the ideal output under perfect conditions. 6) What is downstream processing and why does it have to be considered when calculating the total costs of a bioprocess? Downstream processing=all process steps of product separation and purification after the bioconversion. Currently it is estimated that industrial downstream bioprocessing of biopharmaceuticals constitutes over 40% of the manufacturing cost. The localization of the product strongly influences the downstream processing. The purification of an extracellular product is usually much easier and less costly. Advantages of microtiter plates: When you have a lot of biocatalysts you can find the best one. Important: the same biocatalyst may behave differently in large-scale. 04 ENZYMES AND COFACTORS PART A 1) Recapitulation: enzymes reduce activation energy of a reaction but do not change the equilibrium constant 2) Enzymes stablize the transition state 3) Enzymes are classified by the type of reaction they catalyze; classes of enzymes and EC numbers Note: recently, translocator membrane proteins (catalyze the movement of ions or molecules across membranes) have been considered a seventh class of enzymes (e.g. Vandenberghe et al. 2020). 4) BRENDA is a useful database for obtaining information about enzymes 5) While one group of enzymes act as catalysts solely by its active groups in the active center (to stabilize the transition state), another group of enzymes require co-factors for this task 6) Cofactors can be metal ions or complex organic molecules (many of the latter result from vitamins) 7) Recapitulation: coenzymes and prosthetic groups, examples 8) The difference in the absorption spectrum between NAD(P) and NAD(P)H is widely used for enzyme activity measurements in vitro A coenzyme (organic or metalloorganic molecules) or metal ion that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group (FAD, heme in cytochromes, iron-sulfur proteins, ubiquinone, cannot flow freely in the cytosol, non-soluble) (Note: some coenzymes contain a protein component such as cytochromes or iron-sulfur proteins). NADH or NAD+ are coenzymes, soluble, flow freely in the cytosol (released after the reaction). Cofactors act as transient carriers of specific functional groups. 04 ENZYMES AND COFACTORS PART B 1) What is the unit of specific and volumetric enzyme activity? 2) Enzyme activity can be measured by recording the rate of substrate consumption or product formation 3) NAD+, NADH, NADP+ and NADPH are soluble co-factors and are „consumed“* or „produced“* stoichiometrically with substrate and product, respectively (in enzymatic reactions which depend on one of these co-factors) 4) The difference in the absorption spectrum between NAD(P) and NAD(P)H is widely used for enzyme activity measurements in vitro 5) Understand the concept of the „Optical Test“ (including the question of how to transform absorption of the co-factor into concentration) The „Optical Test“ is a method developed by Otto Warburg in 1936 to measure the activity of NAD(P)-dependent dehydrogenases Coupled enzyme assays – how to measure blood sugar concentration with the indirect help of the „Optical Test“ You see here a famous application of a coupled enzymatic assay: the measurement of blood sugar. Glucose assay with hexokinase and glucose 6-phosphate dehydrogenase. The determination of glucose comprises its phosphorylation catalyzed by yeast hexokinase as an auxiliary reaction which cannot be detected directly. This is coupled to the oxidation of glucose 6-phosphate to 6-phosphogluconate catalyzed by glucose 6-phosphate dehydrogenase (G6PDH). In this second indicator reaction, 1 mol of NADP+ is reduced per mol of glucose 6-phosphate, which in turn is stoichiometrically equivalent to the glucose present in the original sample, to give NADPH, which can be determined spectrophotometrically at 340nm. Hexokinase is an unspecific enzyme that would phosphorylate many hexoses. Since, however, G6PDH is strictly specific for glucose 6-phosphate and would not accept other sugar phosphates, the reaction system is specific for the assay of glucose also in the presence of other carbohydrates. *Note: the co-factors are not really „consumed“ or „produced“; in fact they are oxidized or reduced in enzymatic reactions since they represent electron carriers 05 LARGE-SCALE INDUSTRIAL BIOTRANSFORMATIONS USING INDUSTRIAL ENZYMES 1) What is an industrial enzyme? = Enzymes produced in large amounts and used inindustry Which enzyme class(es) are particularly attractive as industrial enzyme? Hydrolases (EC 3): Break down large molecules into smaller ones using water (e.g., proteases, amylases, lipases, cellulases). Oxidoreductases (EC 1): Catalyze electron transfer reactions (e.g., laccases, peroxidases, alcohol dehydrogenases). Transferases (EC 2): Transfer functional groups between molecules (e.g., transaminases, kinases). Isomerases (EC 5): Rearrange molecular structures without adding or removing atoms (e.g., glucose isomerase). Lyases (EC 4): Break bonds without water or oxidation (e.g., pectin lyases, decarboxylases). Ligases (EC 6): Join two molecules together, often requiring energy (e.g., DNA ligases). Why are extracellular enzymes more attractive than intracellular enzymes? Extracellular enzymes are often more attractive for industrial applications compared to intracellular enzymes due to the following reasons: 1. Easier Recovery and Purification ​ Extracellular enzymes are secreted into the surrounding medium, simplifying extraction. ​ Intracellular enzymes require cell lysis and complex downstream processes to recover them. 2. Reduced Processing Costs ​ The absence of cell disruption steps lowers production costs for extracellular enzymes. ​ Intracellular enzyme recovery involves additional costs for lysis and removal of cellular debris. 3. Higher Stability in Harsh Conditions ​ Many extracellular enzymes are naturally more stable to survive environmental conditions, making them suitable for industrial settings. 4. Continuous Production Feasibility ​ Extracellular enzyme production can be coupled with continuous fermentation systems, enabling easier scalability. ​ Intracellular enzymes are confined to batch processes unless cells are immobilized or permeabilized. 5. Fewer Contaminants ​ Extracellular production often results in cleaner enzyme solutions, reducing impurities compared to intracellular enzyme extracts. 6. Broader Range of Applications ​ Extracellular enzymes are often adapted for degrading complex substrates in the environment, making them versatile for industries like food, textiles, and biofuels. These advantages make extracellular enzymes more practical, cost-effective, and scalable for industrial applications. 2) Examples for large-scale biotransformations using industrial enzymes: - Conversion of starch to glucose - Glucose isomerase: used in production of high fructose syrup (sweetener) - Pectinase(s): a mixture of fungal extracellular enzymes used to facilitate the processing of fruits and vegetables - Lactase: produce lactose-free milk and improve lactose digestability in milk-derived food and feed - Phytase: improve digestibility of animal feed 06 EXAMPLES FOR INDUSTRIAL BIOTRANSFORMATIONS FOR THE PRODUCTION OF CHEMICALS AND PHARMACEUTICALS 1) Examples for important (cell-free and whole cell) biotransformations in the context of producing (bulk and fine) chemicals and pharmaceuticals: - The production of acrylamide (a bulk chemical) using nitrile hydratase - Biotransformations involved in the synthesis of of steroid drugs - The generation of the chiral synthon for the production of atorvastatin (a cholesterol-lowering drug) using enzymes 2) The importance of cofactor recycling systems in biotransformations and how they work ​Cofactor recycling systems are crucial in biotransformations as they ensure the continuous supply of essential cofactors (e.g., NADH, NADPH, ATP) required by many enzymes for catalysis. Since cofactors are expensive and used in small amounts, recycling systems enhance process efficiency, reduce costs, and make biotransformations economically viable. How Cofactor Recycling Systems Work 1.​ Primary Reaction: ○​ An enzyme uses a cofactor (e.g., NADH or NADPH) to drive the desired reaction (e.g., reduction or oxidation). ○​ During the reaction, the cofactor is converted to its oxidized or reduced form. 2.​ Recycling Reaction: ○​ A secondary enzyme regenerates the cofactor to its original form using a cheap substrate. ○​ Example: ​ NADH Recycling: Lactate dehydrogenase converts pyruvate to lactate, regenerating NADH. ​ NADPH Recycling: Glucose-6-phosphate dehydrogenase oxidizes glucose-6-phosphate to regenerate NADPH. Benefits: ​ Reduces the need for expensive cofactor supplementation. ​ Enables continuous biotransformations. ​ Improves yield and sustainability of enzymatic processes. 3) Pros and Cons of using whole cells versus cell-free (isolated) enzymes Advantages: - ​ Bioconversions based on whole-cells containing the required enzyme(s) are generally cheaper and simpler (no separation/purification of enzymes from cells is required). - ​ Enzymes are usually more stable in the long term (since they are protected from the external environment). Disadvantages: - ​ Unwanted by-products might be formed (since in addition to the desired enzyme(s), a multitude of additonal enzymes are present in a whole-cell system. - ​ Lower transformation rates due to mass transfer limitations caused by cell walls and membranes which act as physical barriers and reduce the diffusion of substrates and products (→ this limitation can be overcome by cell permeabilization without affecting the desired enzyme). 07 Non-fossil (Renewable) Substrates for de novo Biosynthesis 1) Non-fossil or renewable substrates (carbon sources for de novo biosynthesis/fermentative processes) contain carbon derived from carbon dioxide and chemical energy from ‘‘present-day’’ conversion of solar energy (e.g. photosynthesis) 2) Non-fossil substrates used in current chemical biotechnology are based on carbohydrates (sugars, starch, lignocellulose) or fats/oils – direct use of CO2 (or other C1 compunds is attractive but challenging – and focus of cutting-edge research) 3) In general, the carbon in non-fossil substrates is less reduced than fossil carbon (= less chemical energy) 4) Sugar (sucrose)-based feedstocks; molasses is a by-product of the sugar extraction from sugar beets and sugar cane 5) Starch-based feedstocks: What is starch? = – a polymer of glucose – is often used as the raw material in order to obtain glucose =a mixture of the polysaccharides amylose (10–30%) and amylopectin (70–90%) both composed of glucose How is it industrially converted to glucose? Enzymatic hydrolysis of granular starch requires gelatinization = the process in which starch becomes soluble, binds water and forms a gel. → can be achieved by heating the starch/water mixture above a certain temperature (about 60-80°C depending on feedstock) 6) Lignocellulose-based feedstocks: What is lignocellulose? Lignocellulose is abundant in plant cell walls and is the generic term for the complex construct of three polymers (cellulose, lignin, hemicelluloses). It makes the cell wall rigid, protects the cell and plant. Protective function. Why is it much more difficult to convert lignocellulosic biomass into metabolizable sugars (in comparison to starch)? What is Lignocellulose? Lignocellulose is a complex, plant-derived material composed of three main components: 1.​ Cellulose: A polysaccharide consisting of long chains of glucose molecules, forming the primary structural framework. 2.​ Hemicellulose: A heterogeneous, branched polysaccharide made of various sugars (e.g., xylose, mannose).  3.​ Lignin: An aromatic polymer that provides rigidity and protects the plant cell wall from microbial attack and enzymatic degradation. Why is it More Difficult to Convert Lignocellulosic Biomass into Metabolizable Sugars Compared to Starch? 1.​ Complex Structure:​ ○​ Lignocellulose is a highly structured, interwoven matrix with cellulose fibers embedded in hemicellulose and lignin, making it physically and chemically resistant to breakdown. 2.​ Lignin Barrier:​ ○​ Lignin acts as a protective shield, hindering enzymatic access to cellulose and hemicellulose. 3.​ Crystalline Cellulose:​ ○​ Cellulose in lignocellulose is highly crystalline and tightly packed, making enzymatic hydrolysis slower and less efficient. 4.​ Heterogeneity of Hemicellulose:​ ○​ Hemicellulose contains a mix of sugars and linkages, requiring a variety of enzymes for complete breakdown, unlike starch, which primarily consists of glucose units. 5.​ Recalcitrance:​ ○​ The combination of physical structure, chemical composition, and lignin makes lignocellulose recalcitrant (resistant) to enzymatic or microbial degradation. 6.​ Pretreatment Requirement:​ ○​ Lignocellulosic biomass requires energy-intensive pretreatment (e.g., chemical, thermal, or biological processes) to disrupt the lignin and hemicellulose matrix before enzymes can act on the cellulose. Comparison to Starch ​ Starch is composed of amylose and amylopectin, which are relatively simple glucose polymers. ​ Starch is amorphous and lacks a protective lignin barrier, allowing direct enzymatic hydrolysis (e.g., using amylases) into glucose, making it much easier and cost-effective to convert into metabolizable sugars. Conclusion The complexity, recalcitrance, and need for pretreatment make lignocellulosic biomass a challenging but valuable resource for sustainable biofuel and biochemical production. 7) Which enzymes are required in order to completely convert 1) starch and 2) cristalline cellulose into glucose? 1) Enzymes Required to Convert Starch into Glucose Starch consists of two components: amylose (linear) and amylopectin (branched). To fully hydrolyze starch into glucose, the following enzymes are needed: 1.​ α-Amylase:​ ○​ Breaks down starch (amylose and amylopectin) into shorter polysaccharides and oligosaccharides by hydrolyzing α-1,4-glycosidic bonds. 2.​ Glucoamylase (Amyloglucosidase):​ ○​ Hydrolyzes α-1,4- and α-1,6-glycosidic bonds in oligosaccharides and maltose, releasing glucose units. 3.​ Debranching Enzymes (e.g., Pullulanase or Isoamylase):​ ○​ Cleave α-1,6-glycosidic bonds at branch points in amylopectin, allowing α-amylase and glucoamylase to act more efficiently. 2) Enzymes Required to Convert Crystalline Cellulose into Glucose Crystalline cellulose is a tightly packed polymer of glucose linked by β-1,4-glycosidic bonds. Complete hydrolysis requires a synergistic action of the following enzymes: 1.​ Endoglucanases (EGs):​ ○​ Cleave internal β-1,4-glycosidic bonds within the cellulose chain, breaking it into shorter cellulose fragments. 2.​ Exoglucanases (Cellobiohydrolases, CBHs):​ ○​ Act on the ends of cellulose chains, releasing cellobiose (a dimer of glucose). 3.​ β-Glucosidase:​ ○​ Hydrolyzes cellobiose and other short oligosaccharides into glucose. Comparison ​ Starch Hydrolysis: Requires fewer steps due to its amorphous and easily digestible nature. ​ Cellulose Hydrolysis: Requires multiple enzyme types working synergistically to overcome its crystalline structure and recalcitrance, making the process more challenging. Both processes rely on precise enzyme mixtures tailored to the substrate for efficient conversion into glucose. 8) What are hemicelluloses? = branched heteropolysaccharides Hemicelluloses are a class of heteropolysaccharides found in plant cell walls, associated with cellulose and lignin. Unlike cellulose, which is a linear polymer made exclusively of glucose, hemicelluloses are branched, amorphous, and composed of a variety of sugar monomers. 9) Pentose sugars (present in hemicelluloses) form a significant portion of the total sugars released from lignocellulosic biomass 10) Glycerol is a waste stream during biodiesel production 08 FUNDAMENTALS OF METABOLISM 1) In order to be able to understand de novo biosynthesis processes, we first need to better understand basics of cellular metabolism and metabolic pathways 2) Catabolism (chemoorganoheterotrophic organisms) provides ATP, reducing equivalents (in the from of NADPH) and 11 principial precursors that are required for anabolism 3) There are two ways how ATP can be formed in cell (SLP and ETP) 1. ​ Substrate-level phosphorylation (SLP) (glycolysis for example, transfer of phosphoryl groups from high energy phosphorylated compounds to ADP). 2. ​ Electron transport phosphorylation (ETP). 4) Several phosporylated compounds whose free energy of hydrolysis is more negative than the one of ATP can be used to generate ATP in substrate level phosphorylation (SLP) 5) Proton motive force (p.m.f.) and chemiosmotic hypothesis – the paradigm for combining electron transport with ATP synthesis in oxidative phosphorylation (ETP) The proton motive force is used to generate ATP via a protein complex called ATP synthase or ATPase. The controlled flux of H+ via the ATPase with their concentration gradient allows the conservation of the energy in the form ofATP. The proton motive force is concerned with two aspects: Chemical concentration (delta pH, inside alkaline). Charge distribution (delta psi, inside negative). The flow of electrons are central to the formation of cellular/chemical energy and it starts with the oxidation of reduced carbon compounds (in chemoorganoheterotrophic organisms). The path of electron flow in metabolism is complex. Electrons move from various metabolic intermediates to specialized electron carriers in enzyme-catalyzed reactions and eventually to a “final electron acceptor”. The term reducing equivalent is commonly used to designate a single electron equivalent participating in an oxidation-reduction reaction. 6) The different roles of the redox couples NAD+/NADH and NADP+/NADPH in catabolism/anabolism Ratio between oxidized/reduced forms: NADP+/NADPH = 0.014 (much more of the reduced form, facilitates reduction of other substrates and oxidation of itself), NAD+/NADH = 700 (much more of the oxidized form, facilitates the oxidation of other substrates and the reduction of itself). Equilibrium of NAD+/NADH supports oxidation of substrates. NAD+ can collect electrons from many substrates. Catabolic use. Equilibrium of NADP+/NADPH supports reduction of substrates. NADPH can reduce many substrates. Anabolic use (synthesis of fatty acids). These two redox couples are the only ones that can float in the cytoplasm (soluble). 7) Amphibolic pathways (such as glycolysis, pentose phosphate pathway, TCA cycle) have catabolic and an anabolic functions 8) The 11 principal precursors for anabolism and the three central metabolic pathways which form these precursors Glycolysis (Glucose 6-phosphate, Triosephosphate, Phosphoglycerate, Phosphoenolpyruvate, Pyruvate) TCA Cycle (Acetyl-CoA, Oxaloacetate, Succinyl-CoA, 2-oxoglutarate) Pentose Phosphate Pathway (provides NADPH) (Pentose phosphate, Tetrose phosphate) 9) Cells require and have to transform energy to perform different forms of cellular work 10) When a reaction proceeds with the release of free energy (i.e. the system changes so as to possess less free energy), the free-energy change (∆G) has a negative value 11) We have to be careful to distinguish between two quantities: the actual free-energy (∆G) change and the standard free-energy change (∆G0 and ∆G0 ); there are defined conditions for the latter 12) The standard free energy change of a chemical reaction (∆G0 or ∆G0‘) is simply an alternative mathematical way of expressing its equilibrium constant (Keq or K‘eq) 13) What are the costs of ATP generation from ADP and inorganic phosphate in cells under standard conditions (∆G0 )? Where does this energy come from? The required standard free energy ∆G0’ is 30.5 kJ/mol (i.e. the opposite sign compared to ATP hydrolysis!) Free energy: ΔG Although in any chemical reaction some energy is lost as heat, in biology we are interested in free energy changes (abbreviated ΔG – ‘Gibbs energy’), which is the energy available to do work. The change in free energy during a reaction is expressed as ΔG and provides information quantifying the difference in energy between the substrates and the products of a reaction. ΔG depends on conditions such as temperature, pressure and concentrations of reactants. ΔG: Actual free-energy change of a reaction under given conditions. ΔG0: Standard free energy-change of a reaction under “standard conditions” (1 atm pressure and 1 M concentrations). ΔG0‘: Standard free-energy change of a reaction under “standard conditions” (1 atm pressure and 1 M concentrations) at pH 7. ΔG0‘ = ΔG0 if no protons are involved in the reaction. All compounds which have a higher free energy of hydrolysis compared to ATP can be used to make ATP in a process called substrate level phosphorylation. - ​ ATP "collects" P-groups from high-energy compounds and may transfer them onto low-energy compounds. - ​ Connection between catabolic (ATP generation) and anabolic pathways (ATP hydrolysis). 09 IMPACT OF METABOLIC ENGINEERING ON DE NOVO BIOSYNTHESIS PROCESSES 1) Cells/living organisms produce a large number of small molecules/chemicals via metabolism but titer, yield and productivity achieved with wild-type cells is often far from sufficient in order to establish a commercial, economically viable production process 2) While a few small molecules/chemicals can be produced by non-recombinant (wild-type) cells/organisms (e.g. ethanol) or random mutants (e.g. glutamate), the science of metabolic engineering has opened completely novel opportunities of targeted engineering of cells for chemicals production 3) What is a metabolic flux and how can metabolic fluxes be modified? Flux (in general) is a vector quantity, describing the magnitude and direction of the flow of a substance. Metabolic flux is the rate of turnover of molecules through a metabolic pathway. The metabolic flux is determined by the enzyme activities involved in a pathway. Rate-controlling steps determine the flux through the entire pathway. Flux, or metabolic flux is the movement of matter through a metabolic pathway. The metabolic flux is regulated by the enzymes involved in a pathway. Within cells, regulation of flux is vital for all metabolic pathways to regulate the pathway's activity under different conditions. Fluxes through different pathways are connected by metabolites and cofactors. 4) What are traditional strain engineering methods? Traditional strain engineering methods for microbial strain development involve modifying microbes to enhance desirable traits such as higher production levels or resistance to certain conditions. These methods include: 1.​ Mutagenesis: ○​ Inducing random mutations using chemicals or radiation to create genetic diversity. 2.​ Selection and Screening: ○​ Identifying and isolating strains with superior traits through systematic testing. 3.​ Adaptive Laboratory Evolution (ALE): ○​ Gradual adaptation of microbes to changing environments by culturing them under selective conditions, enhancing traits that improve growth or productivity. These methods are based on non-directed genetic modifications and rely on natural selection mechanisms, as opposed to metabolic engineering, which involves targeted genetic changes. 5) What is metabolic engineering? Metabolic Engineering: the science of genetically engineering cell‘s metabolism (synonyms: “cellular engineering“/“pathway engineering“). Metabolic engineering: “the improvement of product formation or cellular properties by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology”. Most often, the goal of metabolic engineering is to redirect the metabolic fluxes inside a cell towards the production (and excretion) of a target product with industrial relevance. 6) Know the modifications of enzyme (or transporter) activities which have often been used in metabolic engineering and how they can be achieved at the genetic level In metabolic engineering, enzyme or transporter activities are modified to optimize metabolic pathways and enhance product formation. These modifications and their genetic implementations include: Modifications of Enzyme/Transporter Activities 1.​ Increase in Activity: ○​ Overexpressing an enzyme or transporter by: ​ Increasing the gene copy number. ​ Replacing the native promoter with a stronger one. ○​ Using an alternative coding sequence to achieve higher enzyme activity. 2.​ Reduction or Complete Abolition of Activity: ○​ Reducing enzyme activity by: ​ Replacing the native promoter with a weaker one.  ​ Using a different coding sequence with lower efficiency. ○​ Completely abolishing activity by deleting the gene encoding the enzyme/transporter. 3.​ Establishing New Activities: ○​ Introducing heterologous enzymes or transporters (those not native to the organism) to perform novel functions within the pathway. Genetic Modifications to Achieve These Changes ​ Expression Cassettes: ○​ Designing cassettes with appropriate promoters and ribosome binding sites for prokaryotes or eukaryotes. ○​ Tailoring sequences for efficient transcription and translation in the host organism. ​ Gene Editing and Deletion: ○​ Using technologies like CRISPR-Cas9 or homologous recombination to directly edit or delete genes. ​ Plasmid-Based Expression: ○​ Introducing genes via plasmids that can replicate within the host cell. While this is flexible and allows multiple gene copies, it may require continuous selection pressure for plasmid maintenance. ​ Chromosomal Integration: ○​ Incorporating desired genetic modifications directly into the host chromosome for stability. These strategies allow precise control over enzymatic and transport activities, enabling efficient redirection of metabolic fluxes. 7) Use of plasmids versus chromosomal modifications in metabolic engineering; chromosomal modifications have a number of advantages for stable genetic modifications Advantages of Chromosomal Modifications 1.​ Stability: ○​ Chromosomal integrations are more stable than plasmid-based systems, as they do not rely on external selective pressures (e.g., antibiotics) to maintain the introduced genes. 2.​ No Variation in Gene Copy Number: ○​ Genes integrated into the chromosome are typically present in a single copy (or defined copies in polyploid organisms), ensuring consistent expression levels. 3.​ Long-Term Maintenance: ○​ The modifications are inherited stably by daughter cells during cell division, reducing the risk of gene loss over time. 4.​ No Need for Selection Pressure: ○​ Unlike plasmids, chromosomal modifications do not require the constant application of selection agents to ensure the presence of the genetic modification. 5.​ Reduced Metabolic Burden:  ○​ Plasmid-based expression systems can impose a significant metabolic load on the cell due to replication and maintenance requirements, while chromosomal integration avoids this issue. 6.​ Scalability: ○​ Genetically stable strains are more reliable for industrial-scale applications, where maintaining selective conditions for plasmids is impractical. These advantages make chromosomal modifications a preferred choice for developing robust microbial strains, especially in industrial biotechnology applications Advantages of plasmid-based expression: Multiple copies of plasmid and expressed gene(s). Plasmids are easy to handle/manipulate in vitro. Transformation rates are high. Disadvantages: Plasmid size is limited due to instability. Copy number varies in different strains/cells. Continuous selection pressure necessary. 10 CHROMOSOMAL ENGINEERING 1) Double-strand breaks occur naturally in cells (even though at a low frequency) and have to be repaired. Homologous recombination (HR) is (apart from non-homologous end- joining/NHEJ) a cellular process which allows the repair of DNA double-strand breaks. For the repair of double strand breaks via HR, a repair fragment is required that is homologous to the region of DNA that has to be repaired. 2) To artificially induce double-strand breaks at high frequency at a particular locus in a chromosome (in genetic engineering), several genetic tools have been developed including the genetic scissor CRISPR/Cas9. 3) The process of homologous recombination underlies all targeted modifications conducted in chromosomal DNA: deletions, insertions and replacements 4) You have to be able to explain how the process of homologous recombination is used in order to delete an endogenous gene or insert a heterologous expression cassette in chromosomal DNA. ​Homologous recombination is a natural process that occurs in all cells. It has at least two uses of paramount importance: 1. ​ Repair of chromosomal double-strand breaks. Although double-strand breaks can result from radiation and reactive chemicals, many arise from DNA replication forks that become stalled or broken. 2. ​ Generation of cross-overs during meiosis. Bits of genetic information between two different chromosomes are exchanged to create new combinations of DNA sequences in each chromosome. The potential evolutionary benefit of this type of gene mixing is that it creates an array of new, perhaps beneficial, combinations of genes. The use of homologous recombination machinery for the targeted integration of a particular DNA fragment in one of the chromosomes. We need a double strand break exactly at the position of the chromosome at which we want to integrate a particular DNA sequence. Each genetic locus is naturally affected by random double strand breaks at a very low frequency. Nowadays, we make use of advanced genetic tools (genetic scissors) that are able to introduce double strand breaks at high frequency and exactly at the position at which we want to introduce our DNA fragment (e.g. CRISPR/Cas9). We need to provide the cell with a linear double stranded DNA fragment (= repair fragment) that is homologous to the DNA sequence at the position at which the double strand break is. We can “cheat“ and offer a fragment which is only homologous at the ends, but contains a sequence of our choice in the middle part. 5) Make yourself familiar with the different steps that have to be performed in the lab in order to achieve chromosomal modifications including the verification of correctly integrated DNA by diagnostic PCR. 11 Central Metabolic Pathways – The Respiratory Chain and Oxidative Phosphorylation (PDH COMPLEX AND TCA CYCLE) 1) Oxidative phosphorylation is the culmination of energy-yielding metabolism in aerobic organoheterotrophic organisms 2) Oxidative phosphorylation is one form of electron transport phosphorylation and occurs at the inner mitochondrial membrane (eukaryotes) or at the plasma membrane (bacteria) 3) Electrons from energy-rich reduced compounds enter the respiratory chain via NADH or FADH2 and are eventually transferred to a final electron acceptor (oxygen in aerobic respiration); the electron flow drives the transport of protons against concentration gradient 4) Electrons can only flow from electron carriers with low to high reduction potential 5) Electron carriers function in four membrane-embedded multienzyme complexes that can be physically separated 6) The movement of protons across the membrane is not completely understood. In some cases, the protons are actively pumped by the respective enzyme complex; in other cases, translocation of protons results from the juxtaposition of carriers that accept both electrons and protons (e.g. ubiqinone) with carriers that accept only electrons (e.g. cytochromes) 7) The inner mitochondrial membrane is impermeable for NADH; cytosolic NADH enters the respiratory chain via shuttle systems or via specific NADH dehydrogenases (plants and fungi) 8) Bacterial respiratory chains differ in several aspects from the mitochondrial one and certain bacteria can use final electron acceptors other than oxygen (anaerobic respiration). 9) New definition of the term respiration Respiration can be defined as a biological process in which living organisms convert energy from substrates, such as glucose, into usable forms (e.g., ATP), often by consuming oxygen (aerobic respiration) or using alternative electron acceptors (anaerobic respiration). It involves a series of metabolic pathways, including glycolysis, the citric acid cycle, and the electron transport chain, to release energy stored in chemical bonds. 12 PDH COMPLEX AND TCA CYCLE 1) The citric acid cycle (Krebs cycle, TCA cycle) is a universal central (amphibolic) pathway 2) In catabolism, acetyl-CoA derived from the breakdown of carbohydrates, fats, and proteins are oxidized by the TCA cycle to CO2, with most of the energy of oxidation temporarily held in the electron carriers FADH2 and NADH 3) The connection between glycolysis (pyruvate) and the citric acid cycle is the pyruvate dehydrogenase enzyme complex (located in the mitochondrial matrix) that converts pyruvate and NAD+ to acetyl-CoA, CO2 and NADH by oxidative decarboxylation; the reaction requires three enzymes and five cofactors 4) The role of the TCA cycle is not limited to catabolism, but delivers four- and five-carbon intermediates as precursors for anabolic pathways (e.g. biosynthesis of certain amino acids) 5) The oxaloactetate in the TCA cycle has to be replenished by anaplerotic reactions; there are several options (depending on organism and growth conditions); 6) In certain organisms which are able to grow under anaerobic conditions, the TCA cycle is (or becomes) a branched pathway Important precursors in TCA for anabolism: · ​ Acetyl-CoA · ​ OA · ​ Succinyl-CoA · ​ Alpha ketoglutarate Replenishment of the C4 compounds (mostly OA and Malate) are done via ANAPLEROTIC REACTIONS. Amphibolic pathway: TCA cycle for example. Utilizing both anabolic and catabolic pathways. Citrate is important in human cells, while microorganisms do not depend on the TCA cycle for citrate production. Anaplerotic reactions in the TCA cycle: 1. ​ Pyruvate carboxylase converts pyruvate into OA. Fixes CO2. 2. ​ PEP carboxykinase converts PEP into OA. 3. ​ PEP carboxylase converts PEP into OA (not present in mammals). 4. ​ Malic enzyme converts pyruvate to malate. The branching of the TCA cycle under anaerobic conditions avoids the formation of excess mitochondrial NADH. Under anaerobic conditions, the cells do not have the opportunity to get rid of the NADH formed. They cannot accumulate a lot of NADH without the electrons going somewhere. If this was so, the cycle would stop and there would be no generation of important precursors that the cells would need to form important compounds. Therefore, evolution has allowed for a branched TCA cycle. The cycle goes until Alpha Ketoglutarate from one end (oxidative branch), and from the other pyruvate to OA via anaplerotic rxn, then to malate, fumarate, succinyl-CoA (reductive branch). When oxaloacetate is converted into succinate in the reductive branch, these two reactions consume NADH instead of producing it. 13 SUGAR CATABOLISM AND FERMENTATION 1) The Emben-Meyerhof pathway (glycolysis) is undoubtly the most common pathway of glucose degradation to pyruvate (you have to know all intermediates of glycolysis, as well as the steps where ATP is formed and where NADH is formed, plus the prinicpial precursors) 2) The pathway of glycolysis is an amphibolic pathway since certain principial precursors are delivered (particularly when the enzymes are used in gluconeogenesis) 3) In order to channel sugars other than glucose into the central carbon catabolism, several enzymatic reactions/pathways exist 4) The fate of pyruvate (respiration or fermentation) depends on organism and conditions such as oxygen availability 5) The Crabtree or Warburg effect describes the occurence of fermentation in the presence of oxygen (in baker‘s yeast = Crabtree effect, in cancer cells = Warburg effect) 6) A revised definition of fermentation will be introduced=does not have anything to do with oxygen New definition for the term fermentation in a strict sense: In a fermentative mode, SLP is the exclusive way of generating ATP. The electrons are transferred from the reducing equivalents (NADH) onto carbon intermediates of the metabolism (no electron transport chain is involved in ATP generation). Note that the use of an electron transport chain for ATP generation (ETP) is a characteristic of respiratory metabolism but not of fermentative metabolism. In certain cells/under certain conditions, both modes co-exist: respiro-fermentative metabolism. 7) Several types of fermentation exist but they all have in common that an organic compound (e.g. pyruvate or a derivative of pyruvate) is used as the final electron acceptor for electrons and ETP is NOT involved in ATP generation (only SLP) 14 PENTOSE PHOSPHATE PATHWAY (PPP) 1) Glycolysis, pentose phosphate cycle and TCA cycle are all amphibolic pathways and their role is not restricted to catabolism – these pathways deliver principal precursors for anabolism (apart from ATP and reducing power) 2) Names of the 11 principal precursors and the pathway in which they are generated ​ Glycolysis (Glucose 6-phosphate, Triosephosphate, Phosphoglycerate, Phosphoenolpyruvate, Pyruvate) ​ TCA Cycle (Acetyl-CoA, Oxaloacetate, Succinyl-CoA, 2-oxoglutarate) ​ Pentose Phosphate Pathway (provides NADPH) (Pentose phosphate, Tetrose phosphate) 3) Why does a cell need NADPH (in addition to NADH)? Equilibrium of NADP+/NADPH supports reduction of substrates. 4) The PPP is an amphibolic pathway and delivers NADPH, pentose phosphates and tetrose phosphates. The latter two precursors are required for the biosynthesis of nucleotides, RNA, DNA, and coenzymes (coenzyme A, NADH). 5) The carbon flow through PPP is highly regulated to meet specific cellular needs. 6) Know the different functions of the PPP for the cell (NADPH, certain precursors and/or ATP) Pentose phosphate cycle delivers principal precursors (pentose and tetrose phosphates), NADPH (and sometimes ATP depending on the fate of GA3P) If a cell requires ATP, the pentose phosphate pathway can also be used to generate it by channeling the glyceraldehyde- 3-phosphate to glycolysis 7) In case a cell uses a pentose sugar as the source of carbon, the PPP is required to channel the carbon into the central carbon catabolism 8) The PPP is divided into two parts: 1.) the oxidative part and 2.) the non-oxidative part (what happens during these two parts?) ​ Oxidative phase. o 2 NADPH are formed. Non-oxidative phase (All reactions are reversible, i.e. cells can in principle also form pentose phosphate and tetrose phosphate from C6/C3 without using the oxidative part). o Conversion of C5 to C3 (glyceraldehyde 3 phosphate to glycolysis: energy generation ATP), C6 (regeneration of glucose 6 Phosphate). Transketolases and transaldolases contribute to this phase. 15 GROWTH OF MICROORGANISMS ON CARBON SOURCES WITH 4, 3 OR 2 CARBON ATOMS 1) The lecture scrutinizes the question how cells can survive and grow on carbon sources other than C6 and C5 sugars. Cells can survive and grow on alternative carbon sources besides C6 (hexose) and C5 (pentose) sugars by utilizing specialized metabolic pathways that allow them to metabolize various compounds. These pathways enable the conversion of alternative carbon sources into central metabolites that feed into major metabolic pathways such as glycolysis or the tricarboxylic acid (TCA) cycle. Here are examples of how cells achieve this: 1. Utilization of C1 Compounds ​ Methanol, Formaldehyde, or Methane: ○​ Cells like methylotrophs use the serine cycle, ribulose monophosphate (RuMP) pathway, or calvin cycle to fix C1 compounds into biomass. ○​ Specialized enzymes such as methanol dehydrogenase convert methanol into formaldehyde, which is further assimilated. 2. Utilization of Organic Acids ​ Acetate: ○​ Used by the glyoxylate shunt, bypassing decarboxylation steps in the TCA cycle. ○​ Acetate is converted to acetyl-CoA and integrated into the TCA cycle. ​ Propionate, Succinate: ○​ Fed into the TCA cycle via enzymes like propionyl-CoA carboxylase or succinate dehydrogenase. 3. Utilization of Alcohols and Ketones ​ Ethanol: ○​ Converted to acetaldehyde and acetyl-CoA by alcohol dehydrogenase and acetaldehyde dehydrogenase. ​ Butanol: ○​ Metabolized via oxidation to butyric acid or intermediates entering the TCA cycle. 4. Utilization of Lipids and Fatty Acids ​ Fatty Acids: ○​ Broken down via β-oxidation to generate acetyl-CoA. ​ Triglycerides: ○​ Hydrolyzed into glycerol and fatty acids, which are processed through glycolysis and β-oxidation, respectively. 5. Utilization of Polysaccharides ​ Starch and Cellulose: ○​ Hydrolyzed into glucose or oligosaccharides by extracellular enzymes (e.g., amylases, cellulases). ​ Chitin or Pectin: ○​ Degraded by specialized enzymes to generate monomers feeding into central metabolism. 6. Utilization of Aromatic Compounds ​ Phenolics (e.g., lignin derivatives): ○​ Degraded by monooxygenases or dioxygenases into smaller intermediates (e.g., catechol) and further metabolized in pathways like the β-ketoadipate pathway. 7. Utilization of Nitrogenous Compounds ​ Amino Acids: ○​ Deaminated to produce keto acids that enter the TCA cycle. ​ Urea: ○​ Hydrolyzed to ammonium and carbon dioxide by urease. 8. Adaptation to Complex Carbon Sources ​ Alkanes and Hydrocarbons: ○​ Metabolized by enzymes such as alkane hydroxylases. ​ Complex Waste Substrates: ○​ Microbes utilize metabolic flexibility to degrade and assimilate substrates like glycerol, industrial byproducts, or agricultural residues. Cells achieve these capabilities through genetic regulation, enzyme expression, and sometimes horizontal gene transfer, enabling metabolic flexibility to thrive on diverse carbon sources. 2) Why is growth on C4, C3 and C2 compounds relevant? Which pathways are required to obtain all principal precursors? Cells grow on C4, C3, and C2 compounds by using key pathways like the glyoxylate shunt, gluconeogenesis, and the TCA cycle to produce essential biosynthetic precursors (e.g., pyruvate, acetyl-CoA, and oxaloacetate). These pathways ensure energy generation and the replenishment of intermediates for growth when sugars are unavailable. 3) The degradation of fats (triacylglycerols) results in glycerol (C3) and acetyl-CoA (C2) 4) Other examples for C2 carbon sources are acetate and ethanol 5) Gluconeogenesis is the pathway which allows the formation of all principal precursors when a C4 or a C3 compound is the carbon source. 6) The glyoxylate cycle (present in certain bacteria, fungi and plants, but not in mammals) can replenish the TCA cycle starting from C2 carbon compounds (i.e. it allows growth on ethanol, acetate and fatty acids*) * the catabolism of fatty acids results in the formation of acetyl-CoA (i.e. C2) and the glyoxylate cycle allows the cell to make C4 from C2 and thus replenish the TCA cycle 16 EXAMPLES FOR TRADITIONAL BIOPROCESSES - Part A: Glutamate and Citric Acid 1) Several chemicals have been produced by traditional processes (i.e. the use of the processes date back before the advent of genetic engineering) 2) The production of such compounds by microorganisms and the optimal production conditions have been empirically discovered and optimized L-glutamate is an example for an amino acid traditionally produced in large quantities mainly in C. glutamicum 3) L-glutamate is only secreted under certain conditions and this has led to a hypothesis how feed-back regulation of glutamate biosynthesis is abolished by the particular conditions (the plasma membrane transporter involved in L- glutamate secretion was discovered only recently) 4) Citric acid is the most famous organic acid formed virtually exclusively via de novo biosynthesis mainly by overflow metabolism in filamentous fungi (e.g. Aspergillus species) 5) Extensive citric acid production (and secretion) requires efficient anaplerotic replenishment of the TCA cycle intermediate oxaloacetate Part B: Butanol 1) The ABE (acetone/butanol/ethanol) fermentation found in Clostridia is an example for producing solvents by fermentation 2) ABE fermentation was originally interesting for acetone production (during world war I) but nowadays butanol has received attention as a biofuel; 3) Solventogenic Clostridia are strictly anaerobic bacteria and have a metabolism that solely provides ATP by substrate level phosphorylation (= fermentation in a strict sense) 4) The ABE-fermentation is a two-step process: - first, acids are produced (accompanied to ATP production) and the pH becomes very low (corresponds to the phase when cells grow) - second, the acids are converted to solvents, pH increases (cells do not grow anymore but form spores) 5) ABE fermentation has a number of severe limitations for an economic process Complex and tedious process which is difficult to control Low productivity Butanol toxicity (titer of 20 g/l cannot be exceeded) It is always a mixture of products that is produced- by- products: acetone and ethanol (Low max. theoretical yield: 21 kg butanol/100 kg substrat) Some improvements have been achieved by process engineering: use of higher cell densities In situ product recovery (reduced toxicity)  use of continuous process mode use of immobilized cells (re-use of cells) 6) Nowadays, metabolically engineered organisms have been developed to produce butanol