Microbial Metabolism, Genetics, and Bioengineering Comprehensive Bioengineering Study Guide

Metabolism: Definitions and Functional Categories

Metabolism is defined as all chemical and physical workings of the cell. It encompasses thousands of distinct reactions occurring simultaneously.
Metabolism is divided into two primary general categories:
- Catabolism: This involves the degradation or breaking down of larger molecules into smaller molecules. These reactions are typically exergonic, meaning they involve the releasing of energy.
- Anabolism (Biosynthesis): This involves building larger biomolecules from smaller building blocks. These reactions are typically endergonic and are usually driven by the input of energy harvested from catabolic processes.
Linkage and Complementarity: While anabolism and catabolism have opposite effects, they are fundamentally linked and complementary; a cell cannot maintain one without the other.

Enzyme Action and Structure

Biological Catalysts: Enzymes are biological catalysts that increase chemical reaction rates without being consumed by the reactions or becoming part of the final products.
Necessity for Life: Enzymes do not create reactions—they simply speed them up. While these reactions could occur naturally, they would occur at a rate far too slow to sustain life processes.
Energy of Activation: All chemical reactions require an initial energy input to proceed, known as the activation energy ( $E_a$ ). Enzymes function by lowering the activation energy for specific reactions, allowing them to occur at much faster rates.
Substrate Interaction: An enzyme works on a specific molecule or set of molecules called the substrate(s). The enzyme serves as a physical site where substrates are positioned for interaction.
- The enzyme temporarily binds to the substrate but does not become a product.
- Enzymes can be reused repeatedly.
Enzyme Classification by Composition:
- Simple Enzymes: Consist of protein molecules alone.
- Conjugated Enzymes (Holoenzymes): Contain both protein and non-protein molecules.
- Cofactor: The non-protein portion of a conjugated enzyme. Most inorganic cofactors are metal ions.
- Coenzymes: These are organic co-factors.
- Apoenzymes: The individual protein components that make up a holoenzyme.
Apoenzyme Structure Levels:
- Primary: The linear amino acid chain.
- Secondary: Folded regions (alpha helices and beta-pleated sheets) formed by hydrogen bonds.
- Tertiary: Interacting folded regions forming a stable three-dimensional molecule. This level creates the active site, a specific groove or pocket for substrate binding.
- Quaternary: Multiple folded amino acid chains interacting (observed only in certain complex proteins/holoenzymes).
Induced Fit: A temporary union between the enzyme and substrate occurs when the substrate moves into the active site; the enzyme adjusts its shape slightly to fit the substrate perfectly.

Classification and Control of Enzymes

Classification by Location:
- Exoenzymes: Transported extracellularly to break down large food molecules or harmful chemicals outside the cell.
- Endoenzymes: Retained and function intracellularly.
Classification by Regulation:
- Constitutive Enzymes: Always present and produced in constant amounts regardless of substrate concentration.
- Regulated Enzymes: Production is induced (turned on) or repressed (turned off) in response to changes in substrate concentration.
Types of Enzyme Reactions:
- Synthesis (Condensation) Reactions: Anabolic reactions that form covalent bonds between substrates; these require $ATP$ and release one molecule of $H_2O$ per bond formed.
- Hydrolysis Reactions: Catabolic reactions that break down substrates; these require the input of $H_2O$ to break bonds.
Environmental Sensitivity:
- Enzymes operate within specific ranges of temperature, $pH$ , and osmotic pressure.
- Labile: Chemically unstable enzymes that lose function when habitat conditions change.
- Denaturation: The breaking of weak bonds that maintain the apoenzyme's shape, leading to a loss of function.
Enzyme Inhibition:
- Competitive Inhibition: A substance resembling the normal substrate competes for the active site, blocking the reaction.
- Noncompetitive (Allosteric) Inhibition: Regulatory molecules bind to a site other than the active site (the allosteric or regulatory site). This binding changes the shape of the active site so it can no longer bind the substrate.

Cell Energetics, Redox, and ATP

Energy Management: Cells manage energy through chemical reactions that make/break bonds and transfer electrons.
- Endergonic: Reactions that consume energy.
- Exergonic: Reactions that release energy.
Redox Reactions (Oxidation-Reduction):
- Reduction: The gain of electrons by a molecule.
- Oxidation: The loss of electrons by a molecule.
- Coupling: Oxidation and reduction reactions always occur together. Energy moves with the electrons.
Adenosine Triphosphate (ATP):
- The metabolic "currency" of the cell.
- Structure: Three parts including Adenine (nitrogenous base), Ribose ( $5$ -carbon sugar), and $3$ phosphate groups.
- Energy Release: Removal of the terminal phosphate releases energy for cellular work.
Mechanisms of ATP Formation:
- Substrate-level phosphorylation: Direct transfer of a phosphate group from a phosphorylated substrate to $ADP$ .
- Oxidative phosphorylation: Series of redox reactions occurring during the respiratory pathway.
- Photophosphorylation: $ATP$ formed utilizing sunlight energy.

Bioenergetics and Respiration Pathways

Bioenergetics: The study of cellular energy release mechanisms.
Catabolic Pathways for Glucose:
- Aerobic Respiration: Includes glycolysis, the Krebs cycle, and the respiratory chain. Molecular oxygen ( $O_2$ ) is the final electron acceptor.
- Anaerobic Respiration: Includes glycolysis, the Krebs cycle, and the respiratory chain, but uses oxygen-containing ions (e.g., $NO_3^-$ or $NO_2^-$ ) as the final electron acceptor instead of $O_2$ .
- Fermentation: Uses only glycolysis; organic compounds serve as the final electron acceptors.
Aerobic Respiration Equation: $C_6H_{12}O_6 + 6O_2 + 38ADP + 38P → 6CO_2 + 6H_2O + 38ATP$
Glycolysis Essentials:
- Converts $1$ Glucose ( $6C$ ) into $2$ Pyruvic Acids ( $3C$ ).
- Net yield: $2$ $ATP$ (substrate-level phosphorylation) and $2$ $NADH$ .
Transition Step: Before entering the Krebs cycle, $2$ Pyruvic acids are converted into $2$ Acetyl $CoA$ . This generates $2$ $CO_2$ and $2$ $NADH$ .
Krebs Cycle (TCA Cycle):
- Occurs in the mitochondrial matrix (eukaryotes) or cytoplasm (prokaryotes).
- Acetyl $CoA$ ( $2C$ ) combines with oxaloacetate ( $4C$ ) to form citrate ( $6C$ ).
- Cycle steps: Citrate → Isocitrate → a-ketoglutarate → Succinyl CoA → Succinate → Fumarate → Malate → Oxaloacetate.
- Yield for $2$ turns: $2$ $ATP$ , $6$ $NADH$ , $2$ $FADH_2$ , and $4$ $CO_2$ .
Electron Transport System (ETS) and Chemiosmosis:
- Located in the mitochondria (eukaryotes) or cell membrane (prokaryotes).
- Carriers: NADH dehydrogenase, Coenzyme Q, Cytochrome b, Cytochrome c1, Cytochrome c, Cytochrome a/a3.
- Proton Motive Force: As electrons move, protons ( $H^+$ ) are pumped across the membrane, creating a gradient.
- ATP Synthase: Protons diffuse back through $ATP$ synthase to produce $ATP$ (requires $3$ protons for each $ATP$ ).
- Terminal Step: Oxygen accepts $2$ electrons and $2$ protons to form water: $2H^+ + 2e^- + \frac{1}{2}O_2 → H_2O$ .
- Yield from ETS/Oxidative Phosphorylation: $34$ $ATP$ .
Fermentation Details:
- Incomplete oxidation of glucose in the absence of oxygen.
- Yields small amounts of $ATP$ .
- Products include ethyl alcohol (yeasts) or acids/gases (bacteria) like lactic acid, acetic acid, and propionic acid.

Microbial Genetics: Structure and Function

Genetics: The study of heredity, including trait transmission, expression, variation, and the structure/function and change of genetic material.
Genome: The sum total of genetic material in a cell (chromosomes, mitochondria/chloroplasts, plasmids).
- Bacterial Chromosome: Usually a single circular loop.
- Eukaryotic Chromosome: Multiple and linear molecules.
Genome Sizes:
- Smallest Virus: $4-5$ genes.
- E. coli: Single chromosome, $4,288$ genes, $1 mm$ long ( $1,000 ×$ cell length).
- Human Cell: $46$ chromosomes, $31,000$ genes, $6 feet$ long ( $180,000 ×$ cell size).
Definitions:
- Gene: A fundamental unit of heredity; a segment of $DNA$ containing the code for a protein or $RNA$ .
- Genotype: The genetic makeup (set of genes).
- Phenotype: The observable traits resulting from the expression of the genotype.
DNA Structure (Deoxyribonucleic Acid):
- Double helix comprised of nucleotides.
- Nucleotide Components: $5$ -carbon sugar (deoxyribose), phosphate group, nitrogenous base.
- Bases: Adenine ( $A$ ), Thymine ( $T$ ), Guanine ( $G$ ), Cytosine ( $C$ ).
- Pairing: $A$ binds $T$ ( $2$ hydrogen bonds); $G$ binds $C$ ( $3$ hydrogen bonds).
- Orientation: Anti-parallel strands ( $3'$ to $5'$ and $5'$ to $3'$ ).

DNA Replication

Semi-conservative Replication: Each new chromosome contains one parent strand and one new strand.
Enzymes Involved:
- Helicase: Unwinds and unzips the double helix.
- Primase: Synthesizes an $RNA$ primer.
- DNA Polymerase III: Adds nucleotides in a $5'$ to $3'$ direction.
- DNA Polymerase I: Removes $RNA$ primers and replaces them with $DNA$ .
- Ligase: Links $DNA$ fragments (Okazaki fragments) along the lagging strand.
- Gyrase: Supercoils the replicated $DNA$ .
Strand Synthesis:
- Leading Strand: Synthesized continuously in the $5'$ to $3'$ direction.
- Lagging Strand: Synthesized in short segments ( $5'$ to $3'$ ) called Okazaki fragments; the overall direction is $3'$ to $5'$ .

Protein Synthesis: Transcription and Translation

Central Dogma: $DNA → RNA → Protein$ .
RNA (Ribonucleic Acid): Single-stranded; contains ribose sugar and Uracil ( $U$ ) instead of Thymine ( $T$ ).
RNA Types:
- mRNA (Messenger): Carries the master code in triplets called codons.
- tRNA (Transfer): Has an anticodon that complements the $mRNA$ codon; carries specific amino acids to the ribosome.
- rRNA (Ribosomal): Structural component of ribosomes.
Transcription (Stage I):
- $RNA$ polymerase binds to the promoter region.
- Synthesizes an $RNA$ transcript using the template strand in the $5'$ to $3'$ direction.
- Transcript length: $100-1,200$ bases.
Translation (Stage II):
- Initiation: Ribosome scans $mRNA$ for the start codon (AUG). The first amino acid is Methionine (or formyl-methionine in prokaryotes).
- Elongation: tRNAs bring amino acids to the P and A sites; peptide bonds form.
- Termination: Ribosome reaches a stop codon (UAA, UAG, or UGA) for which no $tRNA$ exists; the ribosome dissociates.
Universal and Redundant Code: The genetic code is used by nearly all organisms, and multiple codons can specify the same amino acid.
Prokaryotic vs. Eukaryotic Differences:
- Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm.
- Eukaryotes: Transcription in the nucleus, translation in the cytoplasm; DNA contains introns (non-coding regions) that must be spliced out by spliceosomes.

Regulation of Gene Expression and Mutations

Operons: Regulatory units in prokaryotes consisting of a regulator, control locus (promoter/operator), and structural locus.
- Inducible (Lac Operon): Normally OFF. Turned on by the substrate (lactose). Lactose (inducer) binds the repressor, causing it to fall off the operator.
- Repressible (Arg Operon): Normally ON. Turned off by product accumulation (arginine). Arginine binds to the repressor, enabling it to bind the operator and block synthesis.
Mutations: Changes in the nitrogen base sequence.
- Spontaneous: Errors in replication without a known cause.
- Induced: Caused by exposure to mutagens (radiation/chemicals).
Categories of Point Mutations:
- Missense: Changes a single amino acid.
- Nonsense: Changes a normal codon into a stop codon (usually severe).
- Silent: Altered base does not change the amino acid.
- Back-mutation: Mutated gene reverses to original sequence.
- Frame-shift: Addition or deletion of bases alters the entire reading frame (usually results in nonfunctional proteins).

Genetic Transfer and Engineering

Genetic Recombination:
- Conjugation: Direct transfer via pilus. Donor ( $F^+$ ) transfers a plasmid to recipient ( $F^-$ ).
- Transformation: Indirect transfer; recipient cell accepts $DNA$ fragments from a lysed donor cell.
- Transduction: Bacteriophage carries $DNA$ from a donor to a recipient.
Transposons: "Jumping genes" that shift locations within the genome.
Recombinant DNA Technology: Deliberate modification of an organism's genome for industrial or medical use.
- Cloning: Inserting a target gene into a vector (plasmid/virus) and then into a cloning host (bacteria/yeast).
Genetically Modified Organisms (GMOs):
- Medical Products: Insulin, Human Growth Hormone ( $HGH$ ), Factor VIII, Erythropoietin ( $EPO$ ), Interferons.
- Plants: Purple cherry tomatoes (high antioxidants), Wheat (fungal resistance), Golden rice (beta-carotene).
- Animals: Pig (Factor VIII), Sheep (alpha antitrypsin), Goat (spider silk in milk).
Gene Therapy:
- Ex Vivo: Cells removed, treated with a healthy gene, and reinserted.
- In Vivo: Naked $DNA$ or vectors introduced directly into the body.

Metabolism is defined as all chemical and physical workings of the cell. It encompasses thousands of distinct reactions occurring simultaneously, facilitating growth, reproduction, structural maintenance, and environmental response.
Metabolism is divided into two primary general categories:
- Catabolism: This involves the degradation or breaking down of larger molecules into smaller molecules. These reactions are typically exergonic, meaning they involve the releasing of energy, which can be harnessed to perform cellular work. Common catabolic pathways include glycolysis and the citric acid cycle that break down glucose and fatty acids.
- Anabolism (Biosynthesis): This involves building larger biomolecules from smaller building blocks such as amino acids and nucleotides. These reactions are typically endergonic, requiring energy input that is harvested from catabolic processes. Examples include protein synthesis and DNA replication.
Linkage and Complementarity: While anabolism and catabolism have opposite effects, they are fundamentally linked and complementary; a cell cannot maintain one without the other. This relationship is crucial for the overall energy balance within the cell, as the energy released from catabolic reactions powers anabolic reactions.