Metabolic Engineering: Redirecting Flux Through a Production Host
Designing a production host means supplying the target pathway while preserving the resources the cell needs. This topic uses carbon, ATP and redox balances, competing branches and production control to compare possible designs.
What a learner can do afterwards
- Account for carbon, ATP use or production, and redox-cofactor supply and regeneration in a transplanted pathway
- Identify a competing branch and propose a change that preserves essential host functions
- Compare inducible and growth-coupled production with constant expression, stating their benefits and limits
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Account for carbon inputs and outputs, ATP use or production, and redox-cofactor regeneration. In a carbon-only model, one 5-carbon precursor yielding one 3-carbon product leaves two carbons for two CO2 molecules. Subtract product carbon from input carbon to find the remainder. This does not establish full chemical feasibility. Atom tracing can test the proposed route, as in the previous topic.
At a branch point, a native reaction and the target pathway use the same intermediate. Native consumption can reduce material available for product, but may supply an essential growth precursor. Reducing competing activity or increasing target-pathway capacity may redirect flux. Test growth and product flux after the change. Shared control, substrate supply or another bottleneck can prevent a gain.
For an ATP budget, subtract other cellular needs from ATP production, then divide what remains by the pathway's ATP cost per product. If ATP production is 18 mmol/hour and growth plus maintenance use 10 mmol/hour, 8 mmol/hour remain. A pathway using 2 mol ATP per mole of product has an ATP-budget upper bound of 4 mmol product/hour. Other constraints may lower actual flux.
Apply the same budget method to the carrier the pathway actually uses. With 8 mmol NADPH/hour available after other cellular needs, a cost of 2 mol NADPH per mole of product allows at most 4 mmol product/hour. NADH and NADPH are different carriers. Supply of one does not automatically meet a need for the other. A suitable regeneration or conversion route is needed.
An inducible pathway can be kept low during biomass growth and activated later for production. This may reduce the burden during growth, but does not guarantee higher output. The switching system and production conditions must suit the host. Induction changes when a pathway is expressed; it does not, by itself, make product formation necessary for growth.
Growth coupling means that growth requires positive product flux under specified conditions. It may discourage nonproducing mutants while that requirement holds. It does not, by itself, prove that the fastest-growing cell has the highest product yield. Product flux is a rate; yield is product per amount of feed consumed.
Constant expression can burden growth by using shared resources. If mutants lose a costly pathway and grow faster in the culture, selection can increase their frequency and lower production. Induction may reduce early burden, but mutation and selection can still occur during production. Constant expression can be appropriate when its costs and benefits fit the host.
Account for carbon, ATP and usable cofactor supply. Protect essential branches and compare production controls under the host's stated conditions.
Lesson, questions and flashcards written for this dataset. Metabolism references were studied for facts; their prose was not adapted.
Readings and flashcards written for this dataset. Video references: Khan Academy, Cellular respiration introduction and Overview of cellular respiration. Video licenses have not been verified.
More to read
Testing a feedback explanation
Suppose product output falls while ATP rises. Those observations alone do not identify the cause. The relevant enzyme might respond to ATP, or another change might limit the pathway.
Check whether ATP inhibits that enzyme in this host. Compare enzyme activity at stated ATP concentrations while keeping other conditions fixed. Also distinguish reduced enzyme activity from a reduction in the amount of enzyme present.
If ATP inhibition is supported, include it among the constraints on production. It does not follow that every enzyme slows or that every diversion of carbon to product activates feedback. Changes elsewhere in the network can also affect flux.
Two ways to run without oxygen
Consider two hypothetical hosts operating without oxygen. Host A uses a respiratory electron transport chain with nitrate as its terminal electron acceptor. Host B regenerates NAD+ by reducing pyruvate to lactate, without a respiratory electron transport chain in that process.
A performs anaerobic respiration. B uses fermentation. In B, NAD+ regeneration lets glycolysis continue supplying ATP. The lactate-forming reaction itself does not make ATP.
For a production design, identify how the host obtains ATP and regenerates its carriers under the proposed conditions. Carbon balance alone cannot answer those questions. Oxygen absence does not force every microbe into fermentation, and fermentation routes need not produce the same products.
Protecting growth at a competing branch
Map the uses of the target precursor before changing a competing branch. A native route may supply a byproduct, an essential growth precursor, or both. Record which outputs the host still needs.
After reducing competing activity, measure growth as well as product formation. Sending a larger fraction of carbon to product may not improve total production if too little biomass remains. If the shared intermediate accumulates, investigate whether a later step limits its use.
Enzyme abundance is not a measurement of flux. Substrate availability, regulation and other reactions also affect the result. Keep changes supported by measurements under stated conditions rather than assuming that removing a native reaction must help.
A production budget is an upper bound
Suppose a pathway uses one ATP and two NADPH for each product molecule. Calculate each budget separately. Start with ATP production, subtract growth and maintenance demands, then compare the remainder with the product pathway's ATP cost. Do the corresponding calculation for NADPH and its other uses.
If production has 10 mmol NADPH/hour available, the NADPH budget allows at most 5 mmol product/hour: divide 10 by the cost of 2. If only 3 mmol ATP/hour remain, the ATP budget allows at most 3 mmol product/hour for this pathway. Both budgets must hold, so they cannot support more than 3 mmol product/hour together.
This is an upper bound. Transport, enzyme capacity or other constraints may keep the actual flux lower. More carbon feed cannot by itself remove the stated ATP or NADPH limit.
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