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Codon optimization / enzyme active site improvement

Codon optimization / enzyme active site improvement


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Does one necessarily have to use de novo synthesis of DNA when attempting protein expression improvement by codon optimization? Or are there other ways?

e.g. Say the gene coding for the enzyme needed to synthesize a certain secondary metabolite is known & sequenced already. The standard cloning approach is to extract mRNA from (say) plant tissue, then construct a cDNA library & express it in E Coli using a suitable plasmid.

Now if the neucleotide sequence needs to be modified (to increase expression) does one have to start de novo or is there easier ways that can start from the mRNA / cDNA?

A variant of the same question is when I'd want to modify the gene because a protein docking study shows some ways to modify the active site of the enzyme by changing a residue or two in the amino acid sequence.


Kind of an old post, I'm surprised it doesn't have an answer yet. There's no reason why you couldn't modify the RNA or cDNA directly, you just have to splice the gene correctly to make the changes you want. You're absolutely right that even a single amino acid change can have a huge difference if it's in the right spot.


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