US 11,739,318 B1
Modified biosensors and biocatalysts and methods of use
Ramesh K. Jha, Los Alamos, NM (US); Charlie E. Strauss, Los Alamos, NM (US); and Taraka Dale, Los Alamos, NM (US)
Assigned to Triad National Security, LLC, Los Alamos, NM (US)
Filed by Triad National Security, LLC, Los Alamos, NM (US)
Filed on Dec. 19, 2018, as Appl. No. 16/226,474.
Claims priority of provisional application 62/607,697, filed on Dec. 19, 2017.
Int. Cl. C12N 15/10 (2006.01); C12N 9/88 (2006.01); C12N 15/52 (2006.01); G01N 33/58 (2006.01); G01N 33/573 (2006.01); C12N 15/74 (2006.01)
CPC C12N 15/1086 (2013.01) [C12N 9/88 (2013.01); C12N 15/52 (2013.01); C12N 15/74 (2013.01); G01N 33/573 (2013.01); G01N 33/581 (2013.01); C12Y 401/0304 (2013.01); G01N 2333/952 (2013.01); G01N 2333/988 (2013.01)] 11 Claims
OG exemplary drawing
 
1. A biosensor comprising:
(i) a protocatechuic acid (PCA) biosensor comprising a nucleic acid encoding a PcaU protein comprising one or more amino acid substitutions at an amino acid corresponding to amino acid position 147, an amino acid corresponding to amino acid position 148 of SEQ ID NO: 56, or both,
wherein the PcaU protein comprises at least 95% sequence identity to SEQ ID NO: 5;
(ii) a cis,cis-muconic acid (ccMA) biosensor comprising a nucleic acid encoding a CatM protein comprising an amino acid substitution at two or more of amino acid positions corresponding to amino acids 97, 127, 128, and 147 of SEQ ID NO: 14;
a CatM-regulated promoter; and
a nucleic acid encoding a reporter protein operably linked to the promoter,
wherein the CatM protein is encoded by a nucleic acid sequence with at least 90% sequence identity to nucleotides 990-1898 of SEQ ID NO: 7; or
(iii) a β-ketoadipic acid (BKA) biosensor comprising a nucleic acid encoding a PcaR protein comprising an amino acid substitution at two or more of amino acid positions corresponding to amino acids 172, 238, 240, and 257 of SEQ ID NO: 19;
a PcaR regulated promoter; and
a nucleic acid encoding a reporter protein operably linked to the promoter,
wherein the PcaR protein is encoded by a nucleic acid sequence with at least 90% sequence identity to SEQ ID NO: 19.