Genetics Chapter on Homologous Recombination

Industrial Biotechnology

• Reflects the status quo of the current chemical industry in terms of sustainability, including raw materials, carbon sources, catalysts, energy consumption, and waste streams.
• White biotechnology is the largest area of biotechnology, focusing on the use of biocatalysts for industrial-scale production and processing of products.
• It aims to reduce the environmental impact of industrial processes and encourage the use of biodegradable polymers and renewable fuels instead of fossil resources.
• Traditional chemical production methods often aren't suitable for renewable carbon sources.
• Biotechnology offers diverse solutions for a sustainable chemical industry, including removing pollutants and preventing future pollution.
• Biocatalyst-based processes are more efficient and environmentally friendly than conventional methods.
• Biocatalysts tend to be self-replicating.

Fossil Carbon

• Carbon stored in fossil fuels (coal, oil, and natural gas) formed from ancient plant and organism remains.
• Fossil carbon represents carbon sequestered over geological time.

Renewable Carbon Sources

• Renewable carbon sources are derived from sustainable and regenerative processes, unlike fossil carbon which is finite and non-renewable.
• Examples include biomass (plant, tree and agricultural crops/organic waste) and biochar (produced via biomass pyrolysis).
• Renewable carbon sources address environmental concerns, including reducing greenhouse gas emissions, promoting circular economy principles, and mitigating climate change.

Enzymes in Industrial Biotechnology

• Enzymes act as catalysts in chemical reactions, either in an isolated form (cell-free) or within cells.
• Industrial biotechnology commonly leverages microorganisms and their enzymes for various applications.

Yeast Saccharomyces cerevisiae

• Yeast Saccharomyces cerevisiae is vital in industrial biotechnology as a cell factory.
• Yeast's features (eukaryote, robustness, Crabtree-positive, facultative anaerobe, extensive metabolic engineering accessibility) contribute to its use in large-scale fermentations.

Biocatalysis

• Biocatalysis is the use of natural catalysts like enzymes or whole cells to perform chemical reactions.
• Biocatalysis is often more specific, efficient, and uses milder conditions than traditional chemical methods.
• Examples of biocatalysis applications include synthesis of pharmaceuticals, production of biofuels, and environmental cleanup.

Biocatalysts

• Biocatalysts encompass any biological molecule or system that accelerates chemical reactions, potentially including isolated enzymes, extracellular enzymes, and whole cells (e.g., microorganisms)

Bioprocesses Part A

• Biotransformation
  • Modifies compounds using biological agents (enzymes/microorganisms), typically to create more valuable/functional products.
• De Novo Biosynthesis
  • Creating complex organic molecules from simple precursors using metabolic pathways.
• Fermentation
  • Uses microorganisms to convert organic compounds (like sugars) into simpler substances in the absence of oxygen, generating energy.
• Whole-Cell Biotransformation
  • Utilizes intact living cells as biocatalysts with metabolic pathways and enzymes for transformations.

Bioprocesses Part B

• Bioreactors offer advantages over smaller vessels like microtiter plates and shake flasks for large-scale bioprocesses (controllable conditions for oxygen, mixing, pH, continuous feeding).
• Different cultivation modes (batch, fed-batch, continuous)
• Batch mode involves a single medium addition; no feeding.
• Fed-batch mode involves adding medium incrementally when substrate levels are low.
• Continuous mode involves constant feeding and product removal to maintain a steady-state.

Product Titer, Yield and Production Rate

• Titer: Concentration of product
• Yield: Ratio of product formed to substrate consumed (g product/g substrate).
• Volumetric Productivity: Conversion rate per unit volume
• Specific Productivity: Conversion rate per unit biomass

Downstream Processing

• Downstream processing (after bioconversion) handles separation and purification of products, which is crucial in evaluating total production costs.
• Extracellular products often have simpler processing compared to intracellular ones, which require cell disruption.

Enzymes and Cofactors Part A

• Enzymes reduce activation energies for chemical reactions without altering equilibrium constants. Enzymes stabilize transition states.
• Enzymes are classified by the type of reaction catalyzed. (Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases).
• EC numbers provide a universal coding system for identifying and classifying enzymes.
• BRENDA is a useful database for obtaining information about enzymes
• Cofactors (coenzymes and prosthetic groups) often required for enzyme function, including vitamins and metal ions.

Enzymes and Cofactors Part B

• Units (U) are the standard units of enzyme activity.
• Optical tests (like coupled enzyme assays) use the characteristics of cofactors (like NAD(P)+/NAD(P)H) absorbance changes to measure enzyme activity quantities.

Large-Scale Industrial Biotransformations Using Industrial Enzymes

• Extracellular enzymes are more often favored over intracellular ones due to easier recovery, reduced costs for production, and higher stability in harsh conditions, as well as continuous production strategies.

Examples of Industrial Enzyme Applications

• Examples include glucose isomerase for syrups, pectinase for fruit processing, lactase for lactose-free products, and phytase for animal feed.

Cofactor Recycling Systems

• Cofactor recycling systems for biotransformations enhance efficiency and reduce costs by enabling the continuous supply of essential cofactors (e.g., NAD(P)H and ATP).
• Secondary enzymes regenerate the initial cofactor using cheap substrates. (e.g., lactate dehydrogenase regenerates NADH)
• Cofactor recycling is crucial for both fermentation and respiration.

Non-Fossil Substrates

• Non-fossil substrates include sugars (e.g., sucrose), starch-based materials using enzymes (a-amylase, glucoamylase, pullulanase), and lignocelluloses (cellulose, hemicellulose, lignin).
• Starch is easier to convert into glucose than lignocellulose due to structural differences in the carbohydrate molecule.

Metabolic Engineering

• Metabolic engineering involves the deliberate modification of a microbe’s metabolism to enhance production pathways, improve efficiency, or adapt under specific conditions, involving use of recombinant technology, and is used in industrial contexts.
• Methods to manipulate enzyme activities include overexpression, activity reduction, and introducing heterologous enzymes.

Chromosomal Engineering

• A strategy used to modify microbial genetic material for targeted modifications (deletions, insertions, replacements) requiring homologous chromosomes, including use of tools (like CRISPR/Cas9) used to induce targeted double strand breaks.
• Chromosomal integration offers advantages for production, stability (reducing the need of continuous selection), and scalability, which is vital for industrial applications.

Central Metabolic Pathways: Respiratory Chain and Oxidative Phosphorylation

• Electrons from energy-rich compounds enter the respiratory chain.
• Electron flow drives proton transport (for creating the proton motive force/electrochemical gradients/energy currency in cells) ultimately involving electron donation from organic molecules to oxygen in aerobic respiration).
• The proton motive force is used to synthesize ATP (via ATPase/ATP synthase).

Cellular Metabolism and Fermentation

• Fermentation is a metabolic process that does not require oxygen but utilizes an organic compound as a terminal electron acceptor.
• This process generates energy through substrate-level phosphorylation by directly transferring phosphate from a phosphorylated intermediate to ADP.
• Both fermentation and respiration pathways work in concert, depending on available substrates and environmental conditions.

Pentose Phosphate Pathway (PPP)

• The pentose phosphate pathway (PPP) is an anabolic/catabolic pathway, generating intermediates, reducing power (NADPH), and ATP.
• The pathway is two-phased (oxidative and non-oxidative).
• Provides precursors for nucleotide and amino acid biosynthesis.

Growth of Microorganisms on Different Carbon Sources

• Cells can adapt to different carbon sources, such as C1 compounds (e.g., methanol).
• Specialized enzymes are used for handling different carbon substrates, which are then often funneled into central metabolic pathways (e.g., TCA cycle) for energy production and synthesis of biomass compounds.
• Utilization of diverse carbon sources is particularly crucial for the industrial use of microorganisms for chemical production.

Test your knowledge on the mechanisms and applications of homologous recombination in cells. This quiz covers topics such as double-strand breaks, genetic tools used in research, and the roles of cross-overs in evolution. Perfect for students studying genetics or molecular biology.