Protein engineering for feedback resistance in 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

Abstract

The shikimate pathway delivers aromatic amino acids (AAAs) in prokaryotes, fungi, and plants and is highly utilized in the industrial synthesis of bioactive compounds. Carbon flow into this pathway is controlled by the initial enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). AAAs produced further downstream, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), regulate DAHPS by feedback inhibition. Corynebacterium glutamicum, the industrial workhorse for amino acid production, has two isoenzymes of DAHPS, AroF (Tyr sensitive) and AroG (Phe and Tyr sensitive). Here, we introduce feedback resistance against Tyr in the class I DAHPS AroF (AroF_cg). We pursued a consensus approach by drawing on structural modeling, sequence and structural comparisons, knowledge of feedback-resistant variants in E. coli homologs, and computed folding free energy changes. Two types of variants were predicted: Those where substitutions putatively either destabilize the inhibitor binding site or directly interfere with inhibitor binding. The recombinant variants were purified and assessed in enzyme activity assays in the presence or absence of Tyr. Of eight AroF_cg variants, two yielded > 80% (E154N) and > 50% (P155L) residual activity at 5 mM Tyr and showed > 50% specific activity of the wt AroF_cg in the absence of Tyr. Evaluation of two and four further variants at positions 154 and 155 yielded E154S, completely resistant to 5 mM Tyr, and P155I, which behaves similarly to P155L. Hence, feedback-resistant variants were found that are unlikely to evolve by point mutations from the parental gene and, thus, would be missed by classical strain engineering. KEY POINTS: • We introduce feedback resistance against Tyr in the class I DAHPS AroF • Variants at position 154 (155) yield > 80% (> 50%) residual activity at 5 mM Tyr • The variants found are unlikely to evolve by point mutations from the parental gene.

Keywords: DAHP synthase; Enzyme engineering; Feedback resistance; Protein stability; Shikimate pathway.