Fig. 6: Enhancing the feedback regulation ability of PcaQ by rational design.
From: Lignin valorization to bioplastics with an aromatic hub metabolite-based autoregulation system
a The structure of the regulatory protein PcaQ (Uniprot ID: Q0JYV0). b The docking diagram of PcaQ with protocatechuic acid. The protocatechuic acid was predicted to form 2 hydrophobic interactions with residues Met143 and Arg145, and 2 hydrogen bonds with residues Ile216 and Asn144 of the PcaQ. It also formed salt bridges with Arg145. c The docking diagram of mutants PcaQ R145K with protocatechuic acid. The protocatechuic acid, which had the most negative consensus docking score, interacted via 6 hydrogen bonds with the PcaQ R145K including residues Ser115, Asn114, Arg162 and Ile216. It also formed 2 hydrophobic interactions with residues Leu113 and Pro217. d Docking diagram of the mutants PcaQ R145K and N146R with protocatechuic acid. The protocatechuic acid was predicted to form 1 hydrophobic interaction with residues Leu113, and 3 hydrogen bonds with residues Ser115, Asn144, Ile216 of the PcaQ R145K and N146R. It also formed salt bridges with Arg162. Hydrogen bonds, hydrophobic interactions, and salt bridges are indicated in yellow, blue, and red, respectively. e PcaQ and the mutants regulate the transcriptional differences of reporter genes under different concentrations of protocatechuic acid (n = 4). Growth and substance metabolism analysis of H16PQABAD and H16PQMABAD with (f) (3,4-DHBD), (g) (4-HBD) or (h) (VAN) as the sole carbon source in the basal medium. The dark color represents H16PQMABAD, and the light color represents H16PQABAD. Samples were taken at predetermined intervals for assessing growth via OD600nm readings with a cell-free blank. In addition, metabolite concentrations in the media were measured using HPLC-UVVIS (n = 3). Error bars illustrate the standard deviation of three biological replicates. The data are the mean ± SD. Abbreviations: 4-HBD 4-hydroxybenzaldehyde, 3,4-DHBD 3,4-dihydroxybenzaldehyde, VA vanillic acid, VAN vanillin. The genome number of vdh (vanillin dehydrogenase of P. putida KT2440) was PP_3357. The genome number of aldBI (aldehyde dehydrogenase of P. putida KT2440) was PP_0545. The genome number of aldBII (aldehyde dehydrogenase of P. putida KT2440) was PP_2680. Source data are provided as a Source Data file.
