CHE.496/2009/Responses/a11

Metabolic pathway engineering

• Discussion leader: Maria

Rohini's Response

Synthetic Biology for Synthetic Chemistry

• Metabolic engineering- coupling multiple enzymes to create a metabolic pathway which can be manipulated to produce a desired chemical
• Transformational enzymes are not highly expressed because metabolites might not be produced (used to produce desired products)
• Characteristics of a microbial chemical factory:

1) Genetically stable

2) Grow with minimal supplements to the growth medium to minimize production costs

3) Switch on the entire biosynthetic pathway at the correct time in the production process while having the pathway repressed during non-productive growth stages

4) Coordinate the expression of many genes simultaneously

5) CAD system

• Chassis- a stable behaved host cell
• Problem-hosts have the capability to inactivate foreign genes to minimize the metabolic burden of the foreign DNA
• Vector- transfer genetic material into a cell
• Promoters- control gene expression. They present a problem by exhibiting an all-or-none expression where the fraction of cells induced in the culture varies with the inducer concentration.
• Problem with simultaneous engagement of multiple genes: multiple inducers are added to the medium which increase the cost of production and complexity of gene expression timing
• Alternatives:

1) use a non-native RNA polymerase or transcription factor to control the expression of more than one gene

2) group multiple related genes into operons

Bioengineering novel in vitro metabolic pathways using synthetic biology

Assembly of multi-enzyme systems

1) Data generation: create a model that can predict the kinetic parameters

2) Mathematical optimization of the network: carry out a sensitivity analysis on the estimated parameters

3) Semi-rational enzyme manipulation

• Ultimate goal of protein engineering- to be able to generate stable protein folds and catalytically active centers

Rohini Manaktala 20:27, 29 March 2009 (EDT)

Maria

“Bioengineering novel in vitro metabolic pathways using synthetic biology”

• Fermentation works to produce some manufactured chemicals and pharmaceuticals but not all:
  • Some can’t cross the membrane
• Toxic
  • Lox final concentration
• Co-synthesiszed
  • Many bi-products, purification/ isolation is difficult.
• Why are multistep enzyme reactions unfavorable?
  • Assembly of system of enzymes is expensive
  • Dynamic behavior is complex and may include unknown substrate interactions. These interactions are not well understood and move to homeostasis
• Hard to improve
  • Assembly of multi-enzyme systems:
  • Unsustainable at large scales
• Reactions in Bacteria: why are they unfavorable?
  • Normal chemical reactions inside the cell may interfere with the reaction yield
• How can we avoid this?
  • But can inculate by over-expression the target gene
  • Can eliminate unnecessary genes
  • Apply one common tag sequence
  • Eliminate the bacteria- do reactions in cell-free environment
• Need library assembly
• Data: important steps:
  • Model: use diff eqs
• Parameters can be difficult to get: why?
  • Not known if they change with more complexity
  • Unknown max reaction rate
• What to do?
  • Estimate
  • Use methods like mass spectrometry and fast sampling
  • Simpler without cell: easier to stop the reactions and can perturb in many more ways. Reduce cell matrix effects with diafiltration (?)
• Optimize network
  • Sensitivity analysis
• De novo design of enzymes is end goal (in silico)
• Now can design stable folding proteins (was this done in a lab with an amino acid sequence or in a bacteria with a dna sequence?)
  • Constraints:
  • Programs don’t account for configuration change that may happen when ligand binds

“Synthetic Biology for Synthetic Chemistry”

• enzymes may eliminate many steps now needed in synthetic biology
• multiple enzymes can act to purify
• genes encoding enzymes do not need to be over-expressed- why?
  • expressing at too high a level will use unneeded metabolites (nucleotides and aa’s)
  • only need enough to change intermediates at some rate
  • intermediates can be toxic- don’t want them to build up
• why may the existing “standardized parts” not work for a more complex system?
  • b/c expression levels and timing is important
• some parts are patented!
• requirements for mass production:
  • vector should be as stable as possible –why?
  • host ” –why?
  • host should grow with minimal supplements- cost
  • be able to switch on pathway
  • be able to coordinate expression of multiple genes
  • use computer-aided-design
• why can foreign dna be unstable?:
  • combination of transposons, insertion sequence elements, defective phages, integrases, and site-specific recombinases
• make sure all cells have plasmid – how?
  • separate plasmids into each half of daughter cell while dividing (few commercial-level cells have this encoded)
  • kill daughter cells without plasmid
• minimal and uniform copy numbers of plasmids:why?
• low copy numbers will dominate culture
• heterogeneous product=un favorable
• how do we make sure there are correct copy numbers?
  • time the replication of the plasmid with the cell cycle: make sure the copy number has doubled before Division
  • evenly Partition plasmids into daughter cells before
• can depend on size
• ideally expression varies with inducer concentration- not true in reality (digital)
• avoid this?
  • ungroup the expression of the transporter with the inducer control
  • use/make inducer that does not need a transporter to get across the membrane
  • use non-regulated/ nature transporter
• multipule gene:
  • can use different promoters for each: but need multiple inducers
  • use one inducer and have promoters that respond to different strengths

• Maria Fini 02:16, 30 March 2009 (EDT):

Patrick Gildea's Response

• “Bioengineering novel in vitro metabolic pathways using synthetic biology”
  • Chemical routes instead of enzymatic routes prevail in industry because assembly of enzymes is expensive, the biological system is complex and not well understood, and cellular systems always attempt to form homeostasis conditions. Multiple enzyme systems have been designed and built with favorable results – however other circumstances prevent the use of these systems in industry. Now it is possible to apply techniques available today, for development of entire metabolic pathways for use in industrial production of biological chemicals as demonstrated by Keasling. This paper presents an approach to the design of gene systems that code enzymes to produce desired molecules.
  • Figure 2: a diagram of what the design cycle is for designing these systems of enzymes
    • Start out with the assembly of the system (overexpression of required genes, dna synthesis, etc.)
    • collect concentration data on intermediates, products
    • fit a model to the data to predict behaviors
    • determine points in the system where further expression, etc. can be used to increase product
    • adapt the kinetics of system members and loop back to the beginning
  • For experimental modeling of metabolic systems - there are many kinetic parameters for purified proteins, but there is a big lack of data comprising of the kinetics of metabolic pathways (there are a few exceptions to this - E.Coli, yeast, etc. The modeling of the data produced and subsequent analysis of the forces (van der waals, etc.) can be used to predict behaviors and subsequently devise a manner to increase the desired product in the metabolic pathway

• “Synthetic Biology for Synthetic Chemistry”
  • Synthetic biology impacts the development of the components to engineer cellular metabolism and chassis that hosts the chemistry. Metabolic engineering begins with the introduction of genes that encode enzymes which take advantage of locally produced molecules in the chassis metabolic network to produce desired molecules. Enzymes can catalyze in a single step what may require many steps utilizing synthetic chemistry methodologies. Coupling multiple enzymes eliminates the need for purification of intermediates. The Essential Components Required for Chemical Production that are described in the article: Chassis, Vectors, Promoters, Simultaneous Engagement of Multiple Genes, CAD Tools, and Debugging Routines. Last year, the team made use of the first four components. We did use modeling to predict the maximum amount of bioplastic produced but it was not accomplished until the end of the summer. A good example of how synthetic biology can help a large number of people is the case of artemisinic acid produced via E.Coli. The methodology of utilizing the mevalonate pathway in E.Coli is listed in figure 3. The work done by Keasling is metabolic engineering with respect to inserting a gene sequence from a foreign organism. Nowadays, yeast is used as the chassis as opposed to E. Coli because higher production titers are attained with yeast.

• Patrick Gildea 07:18, 30 March 2009 (EDT):

Thaddeus Webb's Response

Novel Metabolic Pathways

This paper outlines the design considerations of designing a synthetic metabolic system

• Fermentation is one way of harnessing metabolic abilities of enzymes but is limited.
• Multienzyme systems are currently made by over expressing recombinant genes of interest
• More data is needed before complete modeling can be done because so far only purified products have been extensively analyzed, not systems.
• Data generation requires rigorous experimental setup. Precise sampling is required.
• Parameter estimation is a challenge when modeling because there are lots of them and it is hard to tell if a problem is parameter based or model structure based.
• Sensitivity analysis is normally used to determine which variables should be tweaked to optimize the system in silico.
• Modeling leads to determination of whether concentrations should be adjusted or if protein function should be altered to optimize the system.
• De novo design of both protein folding and catalytic sites has been demonstrated.

If we choose to design a metabolic pathway the ideas in this paper will be useful.

Synthetic Biology for Synthetic Chemistry

This paper outlines the primary design considerations in synthetic metabolic pathways and demonstrates them with the example of Artemisinin Biosynthesis.

• Don't need to overexpress metabolic proteins because they are only catalytic.
• Need to coordinate steps so a toxic intermediate does not build up.
• Properties of an ideal microbe synthesizer
  • Genetically stable
  • Cheap to grow
  • Have ability to tun off and on the pathway
  • Coordination of genes
  • Easy to design
  • Easy to Debug
• Would like to eliminate the bacterial ability to silence foreign genes.
• Need to make sure vectors will be uniformly passed on and distributed between daughter cells.
• Want even and concentration dependent induction.
• Want cells to grow on cheapest media possible.
• Protein levels will probably be controlled at the level of transcriptional efficiency.
• Functional genomics will provide a means of debugging the system.

Thaddeus Webb 16:44, 30 March 2009 (EDT)