Fluorescent biosensor for acetyl coenzyme A

A recombinant acetyl-coenzyme A (acetyl-CoA) biosensor polypeptide comprising:

• an acetyl-CoA binding protein having an amino acid sequence at least 95% identical to SEQ ID NO:
• 1, wherein the acetyl-CoA binding protein is divided into:
• a first acetyl-CoA binding protein fragment comprising an N-terminal portion of the acetyl-CoA binding protein;
• and
• a second acetyl-CoA binding protein fragment comprising a C-terminal portion of the acetyl-CoA binding protein;
• wherein the first and second acetyl-CoA binding protein fragments collectively include all of the amino acids of the acetyl-CoA binding protein;
• and
• a fluorescent protein inserted between the first and second acetyl-CoA binding protein fragments and attached to a C-terminus of the first acetyl-CoA binding protein fragment and an N-terminus of the second acetyl-CoA binding protein fragment;
• and
• wherein:
• (i) the C-terminus is an arginine at position 69 of SEQ ID NO:
• 1 (Arg69) and the N-terminus is a glutamic acid at position 70 of SEQ ID NO:
• 1 (Glu70);
• (ii) the C-terminus is a tryptophan at position 23 of SEQ ID NO:
• 1 (Trp23) and the N-terminus is a proline at position 24 of SEQ ID NO:
• 1 (Pro24);
• (iii) the C-terminus is a valine at position 71 of SEQ ID NO:
• 1 (Val71) and the N-terminus is a threonine at position 72 of SEQ ID NO:
• 1 (Thr72);
• (iv) the C-terminus is an aspartic acid at position 99 of SEQ ID NO:
• 1 (Asp99) and the N-terminus is an alanine at position 100 of SEQ ID NO:
• 1 (Ala100);
• (v) the C-terminus is an aspartic acid at 104 of SEQ ID NO:
• 1 (Asp104) and the N-terminus is an arginine at position 105 of SEQ ID NO:
• 1 (Arg105);
• or
• (vi) the C-terminus is a glycine at position 116 of SEQ ID NO:
• 1 (Gly116) and the N-terminus is a phenylalanine at position 117 of SEQ ID NO:
• 1 (Phe117);
• and
• wherein the recombinant acetyl-CoA biosensor polypeptide selectively binds acetyl-CoA, and the binding of acetyl-CoA induces a change in the fluorescence of the fluorescent protein.