Mutants of Micromonospora viridifaciens sialidase have highly variable activities on natural and non-natural substrates

Abstract

This study aimed to improve the hydrolase activity of the well-characterised bacterial sialidase from Micromonospora viridifaciens. The enzyme and its mutated versions were produced in Bacillus subtilis and secreted to the growth medium. Twenty amino acid positions in or near the active site were subjected to site-saturation mutagenesis and evaluated on the artificial sialidase substrate 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid and on the natural substrate casein glycomacropeptide. A considerably higher fraction of the mutants exhibited increased activity on the artificial substrate compared with the natural one, with the most proficient mutant showing a 13-fold improvement in kcat/Km. In contrast, no mutants displayed more than a 2-fold increase in activity on the natural substrate. To gain further insight into this important discrepancy, we analysed the stability of mutants using the PoPMuSiC software, a property that also correlates with the potential for introducing chemical variation, after validating the method with a set of experimental stability estimates. We found a significant correlation between improved hydrolase activity on the artificial substrate and reduced apparent stability. Together with the minor improvement on the natural substrate this shows an important difference between naturally evolved functionality and new laboratory functionality. Our results suggest that when engineering sialidases and potentially other proteins towards non-natural substrates that are not optimized by natural evolution, major changes in chemical properties are advantageous, and these changes tend to correlate with decreased stability, partly explaining commonly observed trade-offs between stability and proficiency.

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria

“…For L. lactis we chose to optimize expression of a tyrosine ammonia lyase (TAL) from Flavobacterium johnsoniae , which is expressed in the cytoplasm and converts tyrosine into p -coumaric acid in a single enzymatic step [ 29 ]. For B. subtilis we chose to target a Micromonospora viridifaciens sialidase (SIA), a hydrolase that cleaves the sialic acid residues of glycans [ 30 ]. In both cases well-established expression set-ups were used that were reported to result in high expression levels.…”

“…We first integrated the selection module hp- Cm R downstream of the genes-of-interest into the plasmids used in the previous studies for the corresponding enzyme production [ 29 , 30 ]. In these experiments, cytoplasmic expression of tal in L. lactis was controlled by the inducible P nisA promoter in a pNZ8048 vector; however, we had to exchange the original chloramphenicol cassette of the vector backbone for an erythromycin resistance gene, to be able to first select for the transformed plasmid then utilize the hp - Cm R device for selection of highly expressing variants.…”

“…We chose to optimize TAL in L. lactis , due to the wide range of compounds of biotechnological interest that are produced from the intermediate p-coumaric acid; these include for example the flavonoid naringenin or the stilbene resveratrol [ 11 , 29 ]. For B. subtilis we optimized the production of a sialidase, as their enzymatic activity gained interest in relation to treatment of spinal cord injuries and there was a recent attempt to improve their activity and production using B. subtilis [ 30 ]. Both genes were cloned into vectors using traditional restriction enzyme based cloning and were already expressed with high yield.…”

“…The replicative vector pDP66K-SIA-hp-Cm R was constructed by adding the hp-Cm R module to the plasmid pDP66K-Mv [ 30 ] which uses the promoter P32 to drive transcription.…”

“…Library construction was performed as described before [ 14 ]. The reference construct for the sia gene, here referred to as the TIR orig clone, was previous described [ 30 ].…”