Question 1. (a) Metabolic engineering has yielded a yeast strain capable of making cannabidiolic acid (CBDA) when hexanoic acid is present in the growth medium. This was accomplished by modifying the wild type gene encoding erg20 and introducing four new enzymes (polyketide synthase, cyclase, CsPT4 and CBDAS) from Cannabis sativa (indicated in the following scheme).

Briefly explain why the enzyme erg20 in the yeast strain, which catalyses the conversion of GPP into farnesyl pyrophosphate (FPP), had to be modified to lower its activity.

(b) Naturally occurring yeast strains express an enzyme that converts hexanoic acid to hexanoyl- CoA. Olivetolic acid, however, is made by the polyketide synthase (a), which uses malonyl-CoA and hexanoyl-CoA as substrates, and the cyclase (b). Draw the product formed by polyketide synthase

(a) and give an example of a reaction used in its formation (a curly arrow mechanism is not required).

(c) Write a curly-arrow mechanism for the CsPT4-catalysed reaction given that Mg2+ ions are required for activity.

(d) The engineered yeast strain was found to produce 4.3 μg/L of CBDA when 1.1 mM hexanoic acid was added to the growth medium. Propose two strategies that might increase the yield of CBDA and briefly justify your answer.

Question 2. (a) Consider the following reaction sequence, which uses traditional synthetic reagents.

Propose a biocatalytic method to carry out the transformation of 1 into compound 2. Your answer should include the enzyme, its required stereoselectivity, any recycling system (if needed) and a curly arrow mechanism explaining the stereochemical outcome.

(b) Propose a biocatalytic reaction, including a suggested enzyme for the conversion of 2 into 3. Explain why the same class of enzyme that you used in part a) cannot be used for converting 2 into 3. A curly arrow mechanism should be provided for your proposed transformation.

(c) Suggest a biocatalytic sequence of your own choosing to generate 1 from simple precursors. Curly arrow mechanisms are not required but you should briefly justify your answer including the stereoselectivity of your chosen enzymes and any recycling requirements.

Question 3. (a) An enzyme E catalyses the following primary metabolic reaction within a bacterium. Note that compound 6 is essential for the survival of the cells.

Draw a curly arrow mechanism for the reaction catalysed by E. You must involve the catalytic residues of the enzyme for full marks. (5 points)

(b) Imagine that you want to make compound 7 on a large scale by biocatalysis but discover that enzyme E does not have suitable substrate selectivity.

Describe a specific series of experiments for directed evolution of E to a descendant capable of catalysing the formation of 7. Your answer should discuss key problems that may be encountered, together with their solutions, and how inactive mutants will be rapidly eliminated. (10 points)

(c) Propose a biocatalytic method for the generation of compound 8 from 7. Your answer should include a curly-arrow mechanism for the enzyme's catalytic reaction and cofactor recycling methodology (if required).