Genetically stable plasmid expressing PDH and FDH enzymes
View Patent ↗Bi-cistronic plasmids used for the expression of formate dehydrogenase (FDH) and modified phenylalanine dehydrogenase (PDHmod) are provided.
1. An isolated nucleic acid molecule encoding a polypeptide having formate dehydrogenase activity and a polypeptide having phenylalanine dehydrogenase activity comprising:
(a) an isolated nucleic acid sequence comprising a nucleotide sequence that is 95% identical to SEQ ID NO: 1; or
(b) a sequence encoding the polypeptide of SEQ ID NO:4 and the polypeptide of SEQ ID NO: 5.
2. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
3. The isolated nucleic acid molecule of claim 1 , wherein the sequence encoding the polypeptide of SEQ ID NO: 4 and the polypeptide of SEQ ID NO: 5 further comprises a single promoter region.
4. An isolated nucleic acid molecule comprising a sequence that hybridizes to the complement of the nucleotide sequence of SEQ ID NO: 1 at 42° C. in a hybridization buffer comprising 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 0.1% SDS, and 10% dextran sulfate, and then at 42° C. in a hybridization wash buffer comprising 0.2×SSC and 0.1% SDS,
wherein the nucleic acid molecule encodes a polypeptide having formate dehydrogenase activity and a polypeptide having phenylalanine dehydrogenase activity.
5. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2.
6. A plasmid comprising the nucleic acid molecule of claim 2 .
7. An isolated host cell comprising the plasmid of claim 6 .
8. The host cell of claim 7 that is a prokaryotic cell.
9. A method for producing (αS)-α-[[(1,1-dimethylethyoxy)carboxyl]-amino]-3-hydroxytricyclo[3.3.1.1 3,7 ]decane-1-acetic acid (Formula I):
wherein the method comprises:
(a) culturing the host cell of claim 8 under suitable conditions to express the polypeptides encoded by the nucleic acid molecule of SEQ ID NO: 1;
(b) partially purifying an enzyme concentrate comprising the polypeptides; and
(c) contacting the culture isolate with an amount of 3-hydroxy-α-oxotricyclo-[3.3.1.1 3,7 ]decane-1-acetic acid (Formula II):
under conditions that allow for production of (αS)-α-amino-3-hydroxytricyclo[3.3.1.1 3,7 ]decane-1-acetic acid (Formula III):
(d) contacting the (αS)-α-amino-3-hydroxytricyclo[3.3.1.1 3,7 ]decane-1-acetic acid with an amount of di-tent-butyl dicarbonate under conditions that allow for production of (αS)-α-[[(1,1-dimethylethyoxy)carboxyl]-amino]-3-hydroxytricyclo[3.3.1.1 3,7 ]decane-1-acetic acid.