Skip to content
📢We are still a growing community and would love some help. Want to contribute? Join us by mailing an e-mail of interest to "r6394175@gmail.com"
Start helping our community →
← Theoretical A

Q11 — Directed Evolution of Enzyme D (NADH-Dependent)

Theoretical A Real exam question — full text reproduced under IBO's CC BY-NC-SA 4.0 license
Recombinant DNA & Biotechnology Techniques

Directed evolution is a process that mimics natural selection by artificially applying selective pressure to organisms to screen and select for a desired phenotype. Directed evolution can be employed in protein engineering.

The Escherichia coli AL strain was engineered by deleting specific pathways that regenerate NAD⁺ (Figure 1). NAD⁺ regeneration can be restored by introducing a foreign enzyme, referred to as Enzyme D, which converts an exogenous substrate, not naturally connected to the native metabolic network, into a non-toxic product using NADH as a cofactor. A library comprising randomized mutants of Enzyme D is introduced into the engineered E. coli AL strain.

Metabolic diagram of the engineered E. coli AL strain: glycolysis feeds into pyruvate, which normally branches to acetyl-CoA and, in the deleted native pathways, to ethanol via acetaldehyde (both consuming NADH to regenerate NAD+). A new heterologous Enzyme D reaction is added, converting an exogenous substrate (labeled A, present in the large intestine) using NADH, regenerating NAD+ in its place. Figure 1. A: exogenous substrate present in large intestine; B: library variants of enzyme D; C: carbon source; D: enzyme D.

On your answer sheet, indicate “T” for true statements and “F” for false ones.

A. Library construction is not required for directed evolution, as Enzyme D can mutate after selective pressure is applied.
B. Enzyme D is under selective pressure only when the strain AL is growing under aerobic conditions.
C. This method of directed evolution allows for differentiating between variants of NADH-dependent enzymes with low activity or high activity, in order to select the variants with the highest activity.
D. E. coli AL strains with variants showing higher activity of Enzyme D have a better chance of survival in the human large intestine.
← Previous: Q10 Next: Q12 →

Question reproduced from IBO 2024, Theoretical Exam Part A, licensed under CC BY-NC-SA 4.0 — attributed to the International Biology Olympiad. Open the full exam PDF · Community solutions (unofficial)