Lecture 9. Engineering Microbial pathways (applications)

Question 1

What are examples of applications of engineered bacteria?

Answer 1

High value chemical production
Diagnostic tools and therapeutics
Engineered Living materials
Bioremediation
Biosensors

Question 2

What are the two broad types of products produced by engineered bacteria?

Answer 2

Chemicals produced by engineered bacteria
Engineered bacteria (live cell)

Question 3

What is the general workflow for metabolically engineering high value chemicals?

Answer 3

1. Project design (What to make?)
2. Selection of a host strain
3. Metabolic Pathway reconstruction
4. Tolerance enhancement
5. Metabolic flux optimisation
6. Scale up

Question 4

What is an example of a small molecule already commercialised and produced by metabolic engineering?

Answer 4

Vanillin

Question 5

Why is hoppy flavour beer without hops important?

Answer 5

Reduces large amount of water used in the beer making process

Question 6

Why are antibiotics derived from microbial sources important?

Answer 6

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

Question 7

How can plastic waste be useful?

Answer 7

Plastic wastes can be biotechnological carbon sources

Question 8

How can synthetic biology be used for CO₂ fixation?

Answer 8

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

Question 9

What are examples of GRAS (Generally Recognised As Safe) species/strains?

Answer 9

B. subtilis, P. putida KT2440, Lactobacillus
thermophilic bacteria, cyanobacteria

Question 10

What is not a GRAS specie but is generally used for tests?

Answer 10

E. coli

Question 11

What are some strategies to enhance tolerance?

Answer 11

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)

Question 12

What is used to identify the clusters in the genome that code for biosynthetic pathways?

Answer 12

AntiSMASH

Question 13

What challenges do heterologous expression of clusters commonly have?

Answer 13

Low production efficiency
Unpredictable gene expression levels (cryptic regulatory elements)

Question 14

What is the most challenging part of metabolic engineering?

Answer 14

Metabolic flux optimisation

Question 15

What is done during metabolic flux optimisation?

Answer 15

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

Question 16

How can live bacteria be detected?

Answer 16

cfu
PCR
ATP assay
Electrophysiology

Question 17

Why is it important to monitor the metabolic engineered system at all time?

Answer 17

If you monitor the outcome you can start doing closed loop approach

Question 18

Why do most drugs have side effects?

Answer 18

Side effects are commonly due to unnecessary exposure. Disease is local but response is global

Question 19

How can side effects from drugs be minimised?

Answer 19

Local delivery

Question 20

How can local delivery of drug be achieved?

Answer 20

1. Environmental sensing
2. Signal integration and decision making
3. Migration to specific tissue
4. Expression of biosynthetic gene clusters
5. Production and delivery of therapeutic molecules
6. Treatment evaluation and tuning
7. Cassis elimination

Question 21

How is environmental sensing influenced?

Answer 21

Specificity, Sensitivity, Dose-dependency

Question 22

What is an example of an engineered bacteria as a therapeutic tool?

Answer 22

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

Question 23

What is an example of engineering bacteria for antigen delivery (vaccination)?

Answer 23

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)

Question 24

What does auxotrophy mean?

Answer 24

Cannot make amino acids, has to rely on the supply from others
Recoding essential genes with non-native amino acid

Question 25

How are engineered bacteria for therapeutic applications contained?

Answer 25

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)

Question 26

What are the challenges of making engineered bacteria for therapeutic applications?

Answer 26

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

Question 27

What are the possible solutions to overcoming the challenges of making engineered bacteria for therapy?

Answer 27

Organ-on-a-chip, computational models, Simulator of the Human Intestinal Microbial Ecosystem (SHIME), Minimise the metabolic burden, Recoding

Question 28

What is organ-on-a-chip?

Answer 28

Microfluidic system where we can culture human cells and mimic the environment of tissues

Question 29

What are examples of engineered-living materials (ELMs)?

Answer 29

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

Question 30

How can bacterial biofilm become conductive?

Answer 30

Can make small region with affinity to gold to have electric conductive material from bacteria

Question 31

How can bacteria be used in concrete?

Answer 31

Microbiome induced calcium precipitation, put bacteria into holes in concrete to fix them

Question 32

What is bio-photo-lithography?

Answer 32

Kill parts of the bacteria you don't want

Question 33

What are the major remaining challenges with ELMs?

Answer 33

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

Question 34

What do bacteria possess like neurones?

Answer 34

Membrane potential

Question 35

What happens in bacteria electrophysiology?

Answer 35

Light interferes with ion channel, keeping it open for a prolonged period, allowing encoded information into the population

Question 36

What occurs in a microbial fuel cell?

Answer 36

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