Recombinantly engineered polypeptides capable of recycling noncanonical cofactors

A recombinantly engineered polypeptide that has improved catalytic efficiency for a reduced form of a noncanonical cofactor,

• wherein the recombinantly engineered polypeptide comprises from 3 to 12 amino acid mutations in comparison to the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof;
• wherein the amino acid mutations promote catalytic efficiency of the recombinantly engineered polypeptide for the reduced form of the noncanonical cofactor and/or reduces catalytic activity for natural cofactor(s), and
• wherein a polypeptide having the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof, has minimal to very limited catalytic efficiency for the reduced form of the noncanonical cofactor;
• wherein the recombinantly engineered polypeptide uses the reduced noncanonical cofactor for cell growth.

A growth selection process to identify recombinantly engineered polypeptides capable of cycling an oxidized form of a noncanonical cofactor, the growth selection process comprising:

• expressing in microorganisms:
• (i) a first recombinantly engineered polypeptide that comprises from 3 to 12 amino acid mutations in comparison to the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof, and which can utilize a reduced form of a noncanonical cofactor to promote microorganism growth;
• and
• (ii) a second recombinantly engineered polypeptide that comprises from 1 to 12 amino acid mutations in comparison to the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof, wherein the introduced amino acid mutations are designed to increase the activity of the recombinantly engineered polypeptide to reduce an oxidized form of the noncanonical cofactor;
• culturing the microorganisms using a first selection criteria to identify variants with enhanced noncanonical-dependent activity, wherein the first selection criteria select for variants that show enhanced noncanonical cofactor activity by exhibiting an increased growth or growth rate in comparison to other transformed microorganisms;
• optionally, culturing the variants using additional selection criteria, wherein the additional selection criteria make it more, and more, challenging for the microorganisms to grow without having catalytic efficiency for the noncanonical cofactor.