KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose

Abstract

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.

KnowVolution of a Fungal Laccase toward Alkaline pH

“…As a result of the balance between screening efforts and gained molecular understanding, KnowVolution enables the tailoring of protein properties with minimal experimental efforts through the integration of directed evolution and computational analysis in four phases. More than ten KnowVolution success stories have been reported for diverse protein properties, for instance, to increase cellobiose activity of an aryl sulfotransferase, to improve the polypropylene binding of a polymer‐binding peptide, or to improve the chain‐length specificity of a hyaluronic acid synthase . Herein, we report the first laccase KnowVolution campaign toward increased tolerance at alkaline pH (pH≥9.0).…”

Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging

“…The true coefficient of variation (CoV) of the screening system is 19.1% (Figure S2a). Screening systems with CoVs < 20% are employed in several successful screening campaigns. ,, After screening of SSM libraries and subsequent rescreening, four variants at positions 189 (E189H, E189 V) and 215 (E215A, E215G) with improved activity (≥1.3-fold) in the SortEvolve assay were identified (Figure S2b). Moreover, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was conducted and confirmed the increased activity of the two selected variants E189H, E215A (Figure S2c).…”

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

“…Various examples of successful KnowVolution campaigns have been published, e. g ., with glucose oxidases, phytases, proteases, cellulases, laccases, aryl sulfotransferases, or polymer‐binding peptides. [ 7 , 30 , 31 , 32 , 33 , 34 ]…”