Synthetic Cell Factories Flashcards
Microbial cell factories
Engineering microbial cells as a platform (factory) to produce or synthesize molecules of interest from feedstocks.
- Sustainability
- Specificity
- Reliability
Specificity
- Traditional chemical synthesis requires lengthy schemes to produce complex molecules
- Specialized (and sometimes expensive, toxic) catalysts are needed to achieve the desired
stereochemistry - Wastage from producing stereoisomeric by-products
- Expressing the pathway enzymes of target compounds enable the bioactive form to be produced with the correct stereochemistry
- Reduced wastage due to by-products with the wrong stereochemistry
- Eliminate the chance of producing toxic diastereomers or enantiomers
- Racemic thalidomide (cancer drug) caused birth defect due to the (S)-isomer
Reliability
Reliability of source for important biochemicals
- Faster production
- Production under controlled environment
- Not susceptible to climatic factors
- Reduce cost of production
Sustainability
- Reduce reliance on fossil resources
- Conversion of renewable feedstocks into useful compounds
- Higher specificity than chemical synthesis
- Create a reliable source for important chemicals
- Reduce energy consumption
- Reduce material cost
- Lower cost of production
Host selection
Safety
* Biological safety level (BSL1), Generally Recognized as Safe (GRAS by FDA), Qualified Presumption of Safety (QPS by EFSA)
Availability of genetic parts and tools
* Promoters, RBSs, terminators, plasmids, etc.
* Genome editing tools
Source of genes
* Prokaryotic or eukaryotic
Inherent properties
* High producer of precursor or product, High tolerance to substrate/product
Common microbial hosts
* Bacteria: E. coli, Lactobacillus spp., Bacillus spp., Yeast: Saccharomyces spp., Yarrowia lipolytica, Kluyveromyces spp., Pichia pastoris
* Unconventional microbes are increasingly being used as production hosts
Pathway design
Overexpress native pathway genes
* Promoter replacement
* Additional gene copies
Heterologous pathway
* Introduce pathway genes from an organism into a selected microbial host
De novo pathways
* Pathways designed by mix-and-matching well-characterized and substrate-promiscuous enzymes from various organisms
Testing
- Growth profile
- Examine the effect of the pathway on cell fitness
- Quantify production level
- Determine the amount of product from each pathway construct
- Metabolomics
- Measure the level of intermediates, native metabolites and by-products produced
- Transcriptomics
- Determine the transcription levels of pathway genes and native genes
- Proteomics
- Measure the protein expression levels of pathway genes and native genes
MCF system
- Identify superior pathway constructs
- Determine the good combinations of promoters, RBSs, etc., that give high production
- Understand the metabolic flux
- Determine the native metabolites that were affected by the pathway
- Detect competing pathways that produce by-products
- Observe the intermediates that accumulated to identify bottlenecks
- Discover physiological changes
- Verify the pathway gene expression
- Determine the changes that the pathway impose on the transcription and protein expression of native genes
- Discover stress response
MCF optimisation
- Re-design pathway construct
- Use stronger/weaker/inducible promoters, RBSs
- Change gene copy number
- Satisfy the needs of the pathway
- Improve co-factor/precursor availability
- Maintain redox balance
- Delete competing pathway genes
- Mitigate stress imposed by the pathway
- Overexpress genes that were down-regulated by the pathway e.g. transcription factor engineering
- Transport product out of the cell
- Regulate pathway expression
- Evolve or engineer strain for enhanced stress tolerance
Production Strain construction methods
- pathway gene fragments
* BioBricks
* Golden Gate
* Gibson
Pathway integration cassette
= production strain with integrated pathway
OR
Pathway plasmid
= production strain with pathway on plasmid
vanillic acid production
Solution
* To decouple cell growth and protein production through the use of biosensors
* Cells would focus on growth first, before enzyme expression
* At nutrient (glucose) rich stage, cells focus on biomass accumulation, without bioconversion
* At nutrient depletion stage, cells switch mode to biotransform substrate into valuable compounds
* Maximize nutrient utilization
* Increased cell growth improves tolerance to the substrates and products
vanillic acid substrate-sensing circuit
- Lignin
Most abundant aromatic polymer on Earth
Constitutes 20-30 % in plants
* Ferulic acid
Major component of lignin
Can be converted to valuable chemicals e.g. vanillic acid
PP3359: Transcriptional repressor, derepressed by feruloyl-CoA
Pech: Feruloyl-CoA-inducible promoter
Reporter: Red fluorescence protein
- Fluorescence was observed upon addition of ferulic acid
- Validated that the substrate sensing circuit functions as intended
vanillic acid substrate and nutrient sensing circuit
- Modified the substrate-sensing circuit for nutrient sensing as well
- Control the fcs gene expression with PcsiD, a carbon starvation inducible promoter that is activated upon nutrient depletion (high cell density)
- Nutrient-sensing unit induced fluorescence at early stationary phase
- Fluorescence induction began later in combination with the substrate-sensing unit
- Validated that the complete circuit is induced by substrate and upon carbon depletion
- Replace the reporter with the pathway enzymes
Substrate and nutrient sensing circuit benefits
- Decouples cell growth and biochemical production through autonomous nutrient-and substrate-sensing
- Improves stress response, growth & productivity
- Achieved ~90% conversion with the complete dynamic controller
Artemisinic acid story background
Artemisinic acid
Precursor to the anti-malaria drug, artemisinin
Artemisinin is traditionally extracted from the plant Artemisia annua
Microbes were engineered to produce artemisinic acid to facilitate production of the anti-malaria drug
Cut price dramatically to benefit developing countries affected by malaria
- Foundation was laid in 2003 using E. coli (ease of manipulation)
- Artemisinic acid biosynthesis pathway genes were not fully identified then
- Artemisinic acid is a terpenoid produced from farnesyl pyrophosphate (FPP)
- ADS was identified from Artemisia annua for biosynthesizing the intermediate amorpha-4,11-diene from FPP
