Industrial Microbiology and Synthetic Biology Study Guide

Introduction to Industrial Microbiology and Learning Outcomes

• Definition: Industrial microbiology is the process of generating useful products utilizing microorganisms. This field intersects with food production, climate change solutions, and various Sustainable Development Goals (SDGs).

• Requirement for Success: Effectively harnessing microbes requires comprehensive knowledge of microbial diversity, structure, physiology, and genetics. This knowledge is essential for:

  • Identifying useful microbial products.

  • Optimizing product output.

  • Ensuring microbial viability within industrial environments.

• Learning Objectives:

  • Identify at least three significant microbial products.

  • Outline methods for discovering and producing natural microbial products.

  • Discuss the role of recombinant microbes in clinical and industrial sectors.

  • Examine techniques for optimizing industrial microbial yields.

  • Apply principles of physiology and gene regulation to industrial scenarios.

Categories and Examples of Microbial Products

Microbial products are broadly categorized into metabolites, proteins, and whole organisms.

1. Metabolites

• Primary Metabolites: These are major products of central metabolism directly linked to growth, produced during the exponential phase. Examples include ethanol and hydrogen.

• Secondary Metabolites: These are minor products made under specific conditions (usually the stationary phase) and are not linked to growth. Examples include antibiotics, vitamins, and alkaloids. These often function in competition or signaling.

2. Proteins

• Enzymes: Used for biocatalysis (e.g., transglutaminase).

• Recombinant Proteins: Used as medicines (e.g., human insulin).

• Nutritional Proteins: Used for food (e.g., mycoprotein).

3. Whole Organisms

• Applications: Used in bioremediation, mining (biomining), or chemical transformations.

• Diatoms: Noted for their unique structures, making them potential platforms for drug delivery.

• Biosensors: Engineered microbes designed to signal the presence of specific chemicals or environmental conditions.

Comprehensive Product Reference Table

Detailed Product Case Studies

Antibiotics (Metabolite Example)

• Produced naturally by soil bacteria for signaling and environmental competition.

• The Streptomyces Genus: Responsible for producing more than $65\%$ of all antibiotics.

• Penicillin: Requires precise nutrient control for maximum yield. The side chain is often chemically modified to create various semisynthetic penicillin derivatives.

Amino Acids (Metabolite Example)

• Produced using regulatory mutants that lack the ability to limit the synthesis of an end product.

• Lysine and Glutamic Acid: Common food industry supplements and flavor enhancers.

Hydrogen (Biofuel Example)

• Highly potent fuel with $3\times$ more potential energy per unit weight compared to gasoline.

• Produced by photosynthetic microbes. Oxygenic photosynthetic microbes store glycogen during the day and use it to fix nitrogen at night, a process that releases $H_2$ gas.

Insulin (Protein Example)

• The first recombinant protein medicine, described in 1979.

• Involves the production of human protein purified from genetically engineered E. coli.

Transglutaminase (Protein Example)

• An enzyme produced by Streptomyces that cross-links the amino acids lysine and glutamine.

• Known colloquially as "meat glue," used to create uniform processed meat products.

Biomining and Plastic Degradation (Whole Microbe Example)

• Acidithiobacillus ferrooxidans: Used to extract copper and gold. It oxidizes pyrite to produce sulfuric acid and $Fe^{3+}$, which then react with the metals for extraction.

• Ideonella sakaiensis: A recently discovered bacterium capable of degrading PET plastic into $CO_2$.

Bioprospecting: Discovering Natural Products

Bioprospecting is the "treasure hunt" for microbial activities in nature. Microbes possess unparalleled diversity, which is exploited when searching for genetic tools like CRISPR-Cas or PCR enzymes.

Roadblocks to Discovery

1. Unknown/Unculturable microbes: The majority of microbes cannot be grown using standard methods.

2. Lack of functional understanding: We often do not know what specific genes do.

3. Silent Genes: Gene clusters (especially for secondary metabolites) that are not expressed under laboratory conditions.solution: Expression in a recombinant host Genetic modification of the original host to drive expression of different genome parts
   
   Solutions for the "Unculturable Majority"

• Specific Culture Conditions: Using anaerobic environments for deep soil microbes.

• The iChip (Microbe Hotel): Growing microbes within their natural environment to provide necessary native factors.

• Genomics: Predicting metabolic requirements or symbiont needs via genome analysis.

• Metagenome Mining:

  • Option 1: Use bioinformatic analysis to identify gene clusters, then express them recombinantly (limited by prior knowledge).

  • Option 2: Systematically clone and express DNA fragments from the environment and screen for functions (requires robust screening methods).

Genetic Engineering and Recombinant Microbes

Recombinant: Possessing DNA from more than one source.

Expression Systems

• Plasmids: Used to control parameters including copy number (via replication machinery), base expression level (via promoter/RBS modification), and specific induction (e.g., using the lac operon induced by IPTG, a stable lactose analog).

Engineering for Yield

• Rational Engineering: Targeted changes such as increasing export systems, removing competing pathways, or deleting repressors to overcome feedback inhibition.

• Random Mutagenesis: Using UV light, transposons, or CRISPR knockouts to create mutations (optimized to average one per cell).

• High-Throughput Screening: Essential for selecting the ideal mutant from thousands using robotic systems.

Specialized Applications: Biosensors

Biosensors repurpose microbial pathways to detect substances and produce readable outputs.

• Genetic (Fluorescent) Biosensor (e.g., Arsenic):

  • Utilizes the ars operon from E. coli.

  • The ArsR regulator and promoter are fused to fluorescent proteins (GFP or mCherry).

  • Cells fluoresce in the presence of arsenic. Output time is approximately $10\,\text{minutes}$.

• Electrical Biosensor (e.g., Thiosulfate):

  • E. coli is engineered with a modified electron transport chain that requires thiosulfate to deliver electrons to an electrode.

  • Produces an electrical signal in response to thiosulfate in approximately $2\,\text{minutes}$.

Industrial Production and Scale-Up

Moving from a flask to industrial levels requires Bioreactors (Fermenters) to provides precise control over temperature, pH, oxidation, and aeration.

Bioreactor Scales

• Lab Scale: $2\,L$

• Pilot Scale: $75\,L$

• Commercial Scale: $1000\,kL$

The Stirred Tank Bioreactor

• Components: Agitator, aeration system, temperature control, and electrodes for pH and dissolved oxygen.

• Operational Goal: Maximize product yield per gram of culture. – Requires understanding of metabolism and growth phases – For primary metabolites, aim to keep cells in exponential phase for as long as possible – For secondary metabolites, prolonging early stationary phase likely to be best

Process Configurations

• Batch Production: Fill, inoculate, grow, and empty.

• Fed-Batch: Adding limiting nutrients (like carbon) to extend the log (exponential) phase.

• Continuous Production: Constant flow of fresh medium and effluent. Theoretically indefinite, but limited by contamination or mutation.

Efficiency and Cost Reduction

• Seed Train: To avoid unproductive lag phases and expensive downtime, microbes are grown in stages (10-50 fold increases) from agar plates to shake flasks, and eventually to the production bioreactor.

• Input Costs: Utilizing byproducts from other industries (e.g., lignocellulose from plant biomass or urea for nitrogen) reduces costs.

Questions & Discussion

• Industrial Lab Tour: Students are encouraged to watch the lecture-associated video featuring Dr. Geoff Dumsday from CSIRO, providing a tour of their industrial microbiology facilities.

• Recommended Reading: Chapters 17 (Microbial DNA Technologies) and 42 (Biotechnology and Industrial Microbiology) of Prescott’s Microbiology, 12th Edition.