Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.
The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains.
Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.
Keya
K
Zhang
Kathryn M
KM
Nelson
Karan
K
Bhuripanyo
Kimberly D
KD
Grimes
Bo
B
Zhao
Courtney C
CC
Aldrich
Jun
J
Yin
D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D061505:Cell Surface Display Techniques / Q000379:methods*; D062385:Hydroxybenzoates / Q000378:metabolism; D009154:Mutation; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D015003:Yeasts / Q000166:cytology / Q000235:genetics*; D062367:ortho-Aminobenzoates / Q000378:metabolism
D062385:Hydroxybenzoates; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C032948:3-hydroxybenzoic acid; C009135:2,3-dihydroxybenzoic acid; D010453:Peptide Synthases