- The figure below depicts a hypothetical scheme for synthesizing a target product in plants. A, B, and C are the precursors of a target product D, whereas E is a by-product. The key enzymes of the pathway are indicated as E1—E6. To enhance the levels of target product D, following strategies were tested:
(A) Enhancing the activity of the enzyme E5 by over- expression and/or protein engineering.
(B) Enhancing the activity of the enzyme E4 by over- expression and/or protein engineering.
(C) Enhancing the levels of C.
(D) Blocking the activity of E6 by RNA-interference or CRISPR/Cas-mediated knockout.
Which of the above mentioned strategies are likely to provide the maximum enhancement of the target product compared to the by-product, if no feedback regulation exists for any of the enzymes in the pathway?
(1) A and B (2) B and C
(3) C and D (4) A and DThe correct answer is (4) A and D.
From the figure: A →(E1)→ B →(E2)→ C →(E3)→ D (target).
From C there is a branch: C →(E5)→ target D and C →(E6)→ by‑product E.
So C is the common precursor; E5 makes product, E6 makes by‑product.
Strategy-by-strategy analysis
(A) Enhancing the activity of enzyme E5 – Helpful
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E5 converts precursor C directly to the desired product D.
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Overexpression/protein engineering that increases E5 activity will channel more C into D, increasing target formation at the branch.
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This clearly raises D relative to E (assuming no feedback).
(B) Enhancing the activity of enzyme E4 – Not optimal alone
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E4 acts upstream (e.g., B → C) and simply raises the pool of C.
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However, C is still partitioned between E5 (to D) and E6 (to E); increasing supply alone will raise flux to both D and E.
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This does not necessarily maximize D relative to by‑product and is less effective than acting at the branch itself.
(C) Enhancing the levels of C – Not optimal alone
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Directly increasing metabolite C again provides more substrate for both E5 and E6.
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Without changing relative activities of E5 and E6, both D and E rise; the by‑product may still consume a substantial share of C.
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So C alone is not the best way to favor D over E.
(D) Blocking activity of E6 (RNAi/CRISPR) – Very helpful
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E6 converts C to by‑product E.
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Knocking out or silencing E6 eliminates the competing sink, forcing most or all available C to flow through E5 into D.
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This both increases D and minimizes E, giving the strongest shift of flux toward the target.
Why A and D together are best
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A (↑E5) increases the capacity of the product‑forming branch.
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D (↓E6) removes the competing by‑product branch.
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Combining them maximizes flux from C into D and minimizes diversion into E, giving the maximum enhancement of product D compared to by‑product E when no feedback regulation is present.
Thus, the correct option is (4) A and D.
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