Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

Abstract

SUMMARY 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 kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km 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.

Engineering polyketide synthases and nonribosomal peptide synthetases

“…Remarkably, this significant change in specificity was achieved via the introduction of only three amino acid substitutions. Yeast surface display was recently used to engineer the specificity of aryl-acid activating A-domains from bacillibactin biosynthesis [45]. Chemically stable bisubstrate analogs of the acyl-AMP adenylate were designed that included non-native structural modifications to the aryl acid portion, in addition to a biotin-linker to enable enrichment of variant A-domains that were able to bind the substrate mimics.…”

Cell surface engineering of industrial microorganisms for biorefining applications

“…Using this system, several proteins, including xylose isomerase (Ota et al, 2013), laccase (Nakanishi et al, 2012), and expansin-like protein , have been displayed on the yeast cell surface. Although numerous technologies for surface display systems have been reported of late (Fushimi et al, 2013;Qiu et al, 2014;Wilde et al, 2012;Yi et al, 2013;Zhang et al, 2013), the development of a wider variety of display systems would broaden the potential applications of surface engineered microorganisms toward industrial applications.…”

Applications of Yeast Surface Display for Protein Engineering

“…In another example, a general strategy for evolving bond-forming enzymes was developed (Figure 4) and applied to identify a bacterial transpeptidase sortase A enzyme with a 140-fold enhancement in catalytic activity 88 . Yeast display has been applied to enhance the activity and/or substrate selectivity of a variety of other enzymes, including firefly luciferase 89 , Rhizomucor miehei lipase 90 , the adenylation domain of a nonribosomal peptide synthetase 91 , E. coli lipoic acid ligase 92 , and a Tobacco Etch virus protease 93 . In general, the success of these strategies was contingent on the enzyme substrate being labeled with an affinity handle or fluorescent probe.…”