Lecture 9. Engineering Microbial pathways (applications) Flashcards
What are examples of applications of engineered bacteria?
High value chemical production
Diagnostic tools and therapeutics
Engineered Living materials
Bioremediation
Biosensors
What are the two broad types of products produced by engineered bacteria?
Chemicals produced by engineered bacteria
Engineered bacteria (live cell)
What is the general workflow for metabolically engineering high value chemicals?
- Project design (What to make?)
- Selection of a host strain
- Metabolic Pathway reconstruction
- Tolerance enhancement
- Metabolic flux optimisation
- Scale up
What is an example of a small molecule already commercialised and produced by metabolic engineering?
Vanillin
Why is hoppy flavour beer without hops important?
Reduces large amount of water used in the beer making process
Why are antibiotics derived from microbial sources important?
Antibiotics derived from microbial sources are rich in chiral centres and hydrogen bond donors and acceptors and span an order of magnitude in molecular weight
How can plastic waste be useful?
Plastic wastes can be biotechnological carbon sources
How can synthetic biology be used for CO₂ fixation?
Conversion of obligate heterotroph to full autotrophy over laboratory timescales
Non-native Calvin cycle operation generates biomass carbon from CO₂ in E. coli
Formate is oxidised by heterologous formate dehydrogenase to provide reducing power
Chemostat-based directed evolution led to complete trophic mode change in ≈200 days
What are examples of GRAS (Generally Recognised As Safe) species/strains?
B. subtilis, P. putida KT2440, Lactobacillus
thermophilic bacteria, cyanobacteria
What is not a GRAS specie but is generally used for tests?
E. coli
What are some strategies to enhance tolerance?
Reduce the intracellular levels of toxic compounds (limit uptake and/or promote export)
Prevent toxic conversion and incorporation to cell biomass
Temporal control of toxic compound production
Adaptive Laboratory Evolution (ALE)
What is used to identify the clusters in the genome that code for biosynthetic pathways?
AntiSMASH
What challenges do heterologous expression of clusters commonly have?
Low production efficiency
Unpredictable gene expression levels (cryptic regulatory elements)
What is the most challenging part of metabolic engineering?
Metabolic flux optimisation
What is done during metabolic flux optimisation?
Gene expression level control (RBS, codon optimisation etc)
Dynamic control (eg oscillation)
Fluxomic analysis
Genome scale metabolic model (GEMs)
Isocost analysis (looking at other proteins)
Culture condition optimisation
How can live bacteria be detected?
cfu
PCR
ATP assay
Electrophysiology
Why is it important to monitor the metabolic engineered system at all time?
If you monitor the outcome you can start doing closed loop approach
Why do most drugs have side effects?
Side effects are commonly due to unnecessary exposure. Disease is local but response is global
How can side effects from drugs be minimised?
Local delivery
How can local delivery of drug be achieved?
- Environmental sensing
- Signal integration and decision making
- Migration to specific tissue
- Expression of biosynthetic gene clusters
- Production and delivery of therapeutic molecules
- Treatment evaluation and tuning
- Cassis elimination
How is environmental sensing influenced?
Specificity, Sensitivity, Dose-dependency
What is an example of an engineered bacteria as a therapeutic tool?
Salmonella typhimurium was attenuated
S. typhimurium colonise on tumours
Arabinose was fed to mice at a right timing. (open loop control)
Tumour weight suppressed when protein expressed
What is an example of engineering bacteria for antigen delivery (vaccination)?
Lactobacillus acidophilus has been engineered to express a B. anthracis protective antigen fused to a dendritic cell-targeting peptide
Engineered B. subtilis spores as vaccine vehicles (engineer proteins on the spores)
What does auxotrophy mean?
Cannot make amino acids, has to rely on the supply from others
Recoding essential genes with non-native amino acid
How are engineered bacteria for therapeutic applications contained?
Auxotrophy
Strict turn on only at the desired location in space and time. (cell density, local signal, logic gate, spatio-temporal control)
Off switch when a chassis leaves its desired location inside the body. (Toxin-AntiToxin system)
What are the challenges of making engineered bacteria for therapeutic applications?
Mutations (Stability, Loss of functions)
Plasmid loss
Inducers (limited spatio-temporal control, safety)
Most circuits are characterised in vitro growth in an ideal medium, an environment far from the realities
What are the possible solutions to overcoming the challenges of making engineered bacteria for therapy?
Organ-on-a-chip, computational models, Simulator of the Human Intestinal Microbial Ecosystem (SHIME), Minimise the metabolic burden, Recoding
What is organ-on-a-chip?
Microfluidic system where we can culture human cells and mimic the environment of tissues
What are examples of engineered-living materials (ELMs)?
Cell-produced functionalised materials (eg polymers, minerals)
Functional living materials formed by cells (unique properties: enzymatic, heat sensitive, conductive)
Self-healing material (e.g. Self-healing microbial concrete)
Dynamic active materials
How can bacterial biofilm become conductive?
Can make small region with affinity to gold to have electric conductive material from bacteria
How can bacteria be used in concrete?
Microbiome induced calcium precipitation, put bacteria into holes in concrete to fix them
What is bio-photo-lithography?
Kill parts of the bacteria you don’t want
What are the major remaining challenges with ELMs?
Production rates and volumes –currently only made at very small scales (~milligram/~litre). The rates of production may be confined by biological time scales.
Long term viability of living organisms
Low-technical barrier technologies to make spatio-temporal patterns
Biocontainment
Economic and Ecological sustainability
What do bacteria possess like neurones?
Membrane potential
What happens in bacteria electrophysiology?
Light interferes with ion channel, keeping it open for a prolonged period, allowing encoded information into the population
What occurs in a microbial fuel cell?
Electroactive bacteria can transfer electrons onto electrodes
They have many applications (e.g. waste water treatment, electric power generation)
Bacteria can dump electrons into metal, generating current
